Egfr antigen-binding molecules and uses thereof

ABSTRACT

Disclosed herein are antigen-binding molecules, such as antibodies, that specifically recognize a portion of the EGFR C-terminal (intracellular) regulatory domain that interacts with one or more regulatory molecules (such as Suppressor of Cytokine Signaling (“SOCS”) proteins). In certain normal or neoplastic cells and/or tissues, this region is inaccessible to the disclosed antigen-binding molecules. Thus, such antigen-binding molecules are useful at least to interrogate the regulated state of EGFR, predict the response of a cancer patient to EGFR inhibitor therapies, and/or predict the aggressiveness of neoplasms.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Nos.60/949,792 filed Jul. 13, 2007 and 60/988,196 filed Nov. 15, 2007,herein incorporated by reference.

FIELD OF THE DISCLOSURE

This disclosure concerns antigen-binding molecules, such as antibodies,specific for a portion of the intracellular domain of epidermal growthfactor receptor (“EGFR”) and uses of such antigen-binding molecules, forinstance, as cancer prognostics and/or as indicators for particular(e.g., EGFR inhibitor) cancer therapies.

BACKGROUND

Cancer is a generic name for a wide range of cellular malignanciescharacterized by unregulated growth, lack of differentiation, and theability to invade local tissues and metastasize. These neoplasticmalignancies affect, with various degrees of prevalence, every tissueand organ in the body. Historically, cancers have been diagnosed usingconventional histological and clinical features of the affected tissueor organ. However, it is now apparent that tumors, even from the sametissue or organ, are heterogeneous on the cellular and/or molecularlevel. As one consequence, the prognosis and/or responsiveness totherapy of each patient may differ. This unpredictability confoundstreatment selection and may expose patients to the risks and discomfortsof unneeded therapies.

EGFR-positive cancers offer a case in point. EGFR and its downstreamsignaling effectors, including members of the Ras/Raf/MAP kinasepathway, play an important role in both normal and malignant epithelialcell biology (Normanno et al., Gene, 366:2-16, 2006). Amplificationand/or mutation of the EGFR gene and/or EGFR protein overexpression havebeen associated with various malignancies, including breast cancer, lungcancer, colorectal cancer, ovarian cancer, renal cell cancer, bladdercancer, head and neck cancer, glioblastoma, and/or astrocytoma.Increased EGFR activity (whether as a result of abnormally high proteinexpression, dysregulation of receptor activity, or other mechanism) isbelieved to contribute to carcinogenesis. Consequently, EGFR is anestablished target for therapeutic development.

Several EGFR inhibitors are available for clinical treatment. Theseinclude EGFR-specific antibodies (e.g., cetuximab (ERBITUX™) andpanitumumab (VECTIBIX™)) and small molecular tyrosine kinase inhibitors(e.g., gefitinib (IRESSA™) and erlotinib (TARCEVA™)). While thesetreatments have benefited subsets of cancer patients, responses to thedrugs are variable. For example, three clinical studies of patients withadvanced colorectal cancer using cetuximab in a monotherapy settingand/or in combination with irinotecan (a chemotherapeutic agent)demonstrated response rates of 10.5% or 10.8% for cetuximab alone and22.5% or 22.9% for the combined therapy (reviewed by Iqbal and Lenz,Cancer Chemother. Pharmacol., 54(Suppl. 1):S32-39, 2004). Similarly,about 10% or about 20% of non-small cell lung cancer (“NSCLC”) patientstreated with 250 or 500 gefitinib per day, respectively, responded tothe drug and exhibited improved symptoms (Birnbaum and Ready, Curr.Treat. Options Oncol., 6(1):75-81, 2005).

Patient responses to EGFR inhibitors have been correlated with variousEGFR metrics. For example, EGFR expression (as measured byimmunohistochemistry) was associated with an objective response toerlotinib treatment in NSCLC patients (Tsao et al., N. Engl. J. Med.,353: 133-144, 2005). However, survival after treatment in these patientswas not influenced by EGFR expression, the number of EGFR copies, orEGFR mutation (Tsao et al., N. Engl. J. Med., 353:133-144, 2005). Inboth preclinical and clinical settings, somatic mutations in the EGFRtyrosine kinase domain were found to correlate with sensitivity of NSCLCpatients to gefitinib and erlotinib but not to cetuximab (Janne et al.,J. Clin. Oncol., 23:3227-3234, 2005). Clinical studies of gefitinibdemonstrated an association between increased EGFR copy number,mutational status, and clinical response in advanced NSCLC (Cappuzzo etal., J. Natl. Cancer Inst., 97:643-655, 2005).

EGFR antibodies in clinical use (e.g., cetuximab (ERBITUX™) andpanitumumab (VECTIBIX™)) bind to the extracellular domain of the EGFR.This receptor domain includes the ligand binding site and theseantibodies are believed to blocking ligand binding; thereby, disruptingEGFR signaling. As a result of the therapeutic utility of such EGFRantibodies, many subsequent studies have focused on the production ofantibodies (or other binding molecules) specific for the EGFRextracellular domain (see, e.g., U.S. Pat. Nos. 5,459,061, 5,558,864,5,891,996, 6,217,866, 6,235,883, 6,699,473, and 7060808; European Pat.Nos. EP0359282 and EP0667165).

Less attention has been paid to antibodies specific for the EGFRcytoplasmic domain, particularly for therapeutic purposes. However, forexample, Hyland et al. proposed the intracellular expression ofsingle-chain antibodies (e.g., scFvs) as a promising approach forselective interference with EGFR signaling. Others have describedantibodies specific for the EGFR intracellular domain at least fordetection purposes (e.g., Lin et al., Cell. Mol. Immunol., 1(2):137-141, 2004; Hyland et al., Oncogene, 22(10):1557-1567, 2003,Panneerselvam et al., J. Biol. Chem., 270(14):7975-7979, 1995; Gullicket al., J. Pathol., 164(4):285-289, 1991; Dazzi et al., Anal. Cell.Pathol., 3(2):69-75, 1991), and some antibodies specific for the EGFRintracellular domain are commercially available (e.g., Epitomics(Burlingame, Calif., USA), Cat. Nos. 1902-1 and 2235-1; Cell SignalingTechnologies (Danvers, Mass., USA), Cat. Nos. 4405 and 2239; SpringBioscience (Fremont, Calif., USA), Cat. No. E2451).

At least one study compared the reactivity of antibodies specific forthe EGFR external and internal domains in the same set of lung cancertissues (Dazzi et al., Anal. Cell. Pathol., 3(2):69-75, 1991). Nosignificant differences in the reactivity of these antibodies wereobserved.

The continued clinical development of EGFR inhibitor therapies wouldbenefit from a parallel strategy for identifying patient populationsmost likely to respond to such treatments. New prognostic and predictivemarkers, which would facilitate an individualization of therapy for eachpatient, are needed to accurately predict patient responses totreatments and help clinicians distinguish among treatment choices forsuch patients.

SUMMARY OF THE DISCLOSURE

Disclosed herein are methods of interrogating the status of EGFRregulation (and, therefore, EGFR activity state) in biological samples(such as formalin-fixed, paraffin-embedded (“FFPE”) tissue sections).EGFR activation status predicts, among other things, the aggressivenessof EGFR-positive neoplasms and/or the potential efficacy ofEGFR-targeted therapies that depend at least in part upon EGFRactivation status. The disclosed methods involve the use ofantigen-binding molecules (also referred to as RD-binding molecules)that specifically bind to the intracellular regulatory domain of EGFR.The status of EGFR regulation in normal or neoplastic cells and/ortissues can be differentiated by the accessibility of the EGFRregulatory domain to RD-binding molecules (including disclosedRD-binding molecules).

Also disclosed are peptides derived from the EGFR regulatory domain(regulatory domain peptides or RDPs) and, in particular, from the regionhaving the sequence LDNPDYQQDFFPKEAKPNG (the “L2G” sequence or peptide;SEQ ID NO: 2). RDPs are useful, at least, in the making of exemplaryRD-binding molecules and as otherwise provided in this disclosure. Insome examples RD-binding molecules, such as antibodies, specificallyrecognize an epitope included in an RDP sequence, for example within theL2G sequence.

The foregoing and other features will become more apparent from thefollowing detailed description of several embodiments, which proceedswith reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a flow chart schematically showing the steps of exemplaryimmunostaining methods.

FIG. 2 is a schematic representation of the EGFR regulatory domainshowing the locations of autophosphorylation sites (white boxes withnumber indicating position of autophosphorylated amino acid residue) andknown binding sites for exemplary EGFR regulatory proteins, includingSignal Transducer and Activator of Transcription (STAT) proteins (e.g.,STAT1 and/or STAT3; OMIM Accession Nos. 600555 or 102582), Growth FactorReceptor-Bound Protein 2 (Grb2; OMIM Accession No. 108355), Signalingand Transforming Protein Containing Src Homology 2 and 3 Domains (Shc)(e.g., OMIM Accession Nos. 600560, 605217, or 605263), Int-P, andSuppressor of Cytokine Signaling proteins (e.g., SOCS1 and/or SOCS3;OMIM Accession Nos. 603597 and 604176, respectively).

FIG. 3 is a series of schematic representations involving the EGFRmolecule which, in its wild-type state, has an extracellularligand-binding domain (LBD) and intracellular tyrosine kinase (TKD) andregulatory (RD) domains. In each schematic, as applicable, EDBMrepresents an extracellular-domain-specific binding molecule (such as anantibody); RDBM represents a regulatory-domain-specific binding molecule(such as an antibody); and RDIM represents a regulatory domaininhibitory molecule (such as a SOCS protein like SOCS1 or SOCS3). Eachof the three schematics (from left to right) represents a differentmolecular setting. The left-most panel shows a full-length EGFR notassociated with a RDIM. The center panel shows a constitutively activeN-terminal truncated EGFR mutant. The right-most panel shows afull-length EGFR associated with a RDIM. In each case, predictions basedon the binding (or not) of the EDBM and RDBM are listed below themolecular schematic.

FIG. 4 shows the results of two Western blots in which three totalprotein concentrations (as indicated) of A431 (EGFR-positive) and BT474(EGFR-negative) cell lysates were run. The left and rightrepresentations show clone 5B7 and clone 3C6, respectively, binding to asingle protein band (appropriate in size for EGFR) in A431 cell lysates.Molecular weights (in kD) are shown at the far left.

FIG. 5 shows the results of staining the indicated tissues with EGFRextracellular-domain-specific clone 3C6 and EGFRregulatory-domain-specific clone 5B7 in tabular (FIG. 5A) and image(FIG. 5B) formats.

FIG. 6 shows images demonstrating the differences in the binding of EGFRregulatory-domain-specific (RDBM) clone 5B7 and EGFRexternal-domain-specific (EDBM) clone 3C6 in two representativenon-small cell lung cancer (NSCLC) tissue sections.

FIG. 7 shows the results of peptide inhibition studies mapping theepitope of the EGFR regulatory-domain-specific clone 5B7. The boxedregion in FIG. 7B represents an exemplary clone 5B7 epitope, theC-terminus of which may be a few amino acids longer or shorter.

FIG. 8 shows images demonstrating the differences in the binding of EGFRregulatory-domain-specific (RDBM) clone 5B7 and EGFRexternal-domain-specific (EDBM) clone 3C6 in two normal mouse livers(left-most two panels) and the livers of SOCS3-knock-out mice(right-most two panels).

FIG. 9 shows two Kaplan Meier plots demonstrating overall survival ofNSCLC patients as a function of clone 3C6 (panel A) or 5B7 (panel B)status. In panel A, 3C6-positive staining is shown by a black line and3C6-negative staining is shown by a gray line. In panel B, 5B7-positivestaining is shown by a black line and 5B7-negative staining is shown bya gray line.

FIG. 10 shows two Kaplan Meier plots demonstrating disease-free survivalof NSCLC patients as a function of clone 3C6 (panel A) or 5B7 (panel B)status. In panel A, 3C6-positive staining is shown by a black line and3C6-negative staining is shown by a gray line. In panel B, 5B7-positivestaining is shown by a black line and 5B7-negative staining is shown bya gray line.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. All sequence database accession numbers referencedherein are understood to refer to the version of the sequence identifiedby that accession number as it was available on the priority date ofthis application (Jul. 13, 2007). In the accompanying sequence listing:

SEQ ID NO: 1 is a reference amino acid sequence (REFSEQ) of human EGFR(isoform a) as set forth in GENBANK™ Accession No. NM_(—)005228. Anucleic acid sequence encoding this polypeptide also is set forth inGENBANK™ Accession No. NM_(—)005228.

SEQ ID NO: 2 is the amino acid sequence of a peptide corresponding toresidues 1167-1185 of SEQ ID NO: 1.

SEQ ID NO: 3 is the curated reference amino acid sequence (REFSEQ) ofhuman Suppressor of Cytokine Signaling 3 (SOCS3) as set forth inGENBANK™ Accession No. NM_(—)003955. A nucleic acid sequence encodingthis polypeptide also is set forth in GENBANK™ Accession No.NM_(—)003955.

SEQ ID NO: 4 is a YXXL/V protein motif

SEQ ID NO: 5 is a YXXP/D protein motif

SEQ ID NO: 6 shows an exemplary RDP consensus sequence.

DETAILED DESCRIPTION I. Introduction

Disclosed herein are EGFR regulatory domain peptides (“RDPs”), whichinclude, e.g., isolated peptides consisting of amino acid residues1167-1185 of SEQ ID NO: 1 or an immunogenic fragment of said peptide.Also disclosed are (EGFR) regulatory domain (RD)-binding molecules thatspecifically binds to such peptides. —Some embodiments include aRD-binding molecule that specifically binds to residues 1138-1196 of SEQID NO: 1 or a SOCS-protein-binding fragment thereof (e.g., aSOCS3-binding fragment).

Also disclosed are compositions including an EGFR RD-binding moleculethe binding of which to EGFR is competitively inhibited by a disclosedRDP (such as an isolated peptides consisting of amino acid residues1167-1185 of SEQ ID NO: 1 or an immunogenic fragment of said peptide).Other disclosed compositions include an EGFR RD-binding molecule thebinding of which to EGFR is competitively inhibited by a Suppressor ofCytokine Signaling (SOCS) protein (such as, a SOCS1 protein (e.g., seeGenBank Accession Nos. NP_(—)003736.1, EAW85163.1 and AAD27709.1) or aSOCS3 protein (e.g., see GenBank Accession Nos. CAG46495.1, CAG38736.1and AAH60858.1), or, in particular embodiments, a SOCS3 protein).

In any embodiment involving a RD-binding molecule (whether compositionor method), an RD-binding molecule can be (but is not necessarily) anantibody (e.g., a monoclonal antibody, such as a rabbit or mousemonoclonal antibody) or a antigen-binding fragment thereof.

Further disclosed are methods of producing an EGFR-specific antibody,comprising immunizing a non-human mammal with an immunogen comprising acarrier protein and a disclosed RDP (such as, amino acid residues1167-1185 of SEQ ID NO: 1 or an immunogenic fragment of said peptide).Some such methods include a further step of isolating serum from thenon-human mammal and isolating polyclonal antibody specific for theimmunogen. Other such methods include a further step of fusing spleencells from the non-human animals with a fusion cell partner to makeantibody-producing hybridomas.

Disclosed methods also include predicting the response of a neoplasm toan EGFR inhibitor by detecting in a biological sample, which includesone or more neoplastic cells, the specific binding of a disclosed EGFRRD-binding antibody to the one or more of the neoplastic cells; whereinspecific binding of the antibody to one or more of the neoplastic cellsindicates that the neoplastic cells will respond to an EGFR inhibitor.In some method embodiments, the neoplastic cell response is slowedgrowth (such as, net zero growth or net negative growth). In othermethod embodiments, the slowed growth is at least 10% (such as at least15%, at least 20%, at least 30%, at least 50%, or at least 75%) lessthan the neoplastic cell growth prior to treatment with the EGFRinhibitor. In some method embodiments, the neoplastic cell response isapoptosis, and, in some such embodiments, at least 10% (such as at least15%, at least 20%, at least 30%, at least 50%, or at least 75%) of theneoplastic cells undergo apoptosis.

Also disclosed are methods for predicting whether a candidate fortreatment with an EGFR inhibitor is likely to respond to such treatmentby detecting in a biological sample from a candidate for treatment withan EGFR inhibitor, which biological sample comprises one or moreneoplastic cells, the specific binding of a disclosed EGFR RD-bindingantibody to the one or more of the neoplastic cells; wherein specificbinding of the antibody to one or more of the neoplastic cells indicatesthat the candidate is likely to respond to treatment with an EGFRinhibitor. In some method embodiments, the specific binding of theantibody to at least 10% (such as at least 15%, at least 20%, at least30%, at least 50%, or at least 75%) of the neoplastic cells in thebiological sample indicates that the candidate is likely to respond totreatment with an EGFR inhibitor.

Other disclosed methods involve predicting the response of a neoplasm toan EGFR inhibitor by detecting in a biological sample comprising one ormore EGFR-positive neoplastic cells substantially no specific binding ofa disclosed EGFR RD-binding antibody to the one or more EGFR-positiveneoplastic cells; wherein substantially no specific binding of theantibody to the EGFR-positive neoplastic cells indicates that theneoplastic cells will not substantially respond to an EGFR inhibitor.Some such methods further involve detecting in a control biologicalmaterial (such as normal skin, normal testis, or normal tonsil) thespecific binding to EGFR of the antibody. Other such methods furtherinvolve detecting in the biological sample specific binding of a secondantibody specific for the EGFR external domain.

Still other disclosed methods involve predicting the response of aneoplasm to EGFR inhibitor administration, by detecting EGFR expressionin a first sample of a biological material comprising one or moreneoplastic cells; and detecting in a second sample of the biologicalmaterial substantially no specific binding to EGFR of a disclosed EGFRRD-binding antibody; wherein detecting EGFR expression in the firstsample and substantially no specific binding to EGFR of a disclosed EGFRRD-binding antibody indicates that the neoplasm is likely to respond toEGFR inhibitor administration. In some such methods, the first sampleand the second sample are serial sections of the biological material.Other such methods further involve detecting in a control biologicalmaterial (such as, normal skin, normal testis, or normal tonsil) thespecific binding to EGFR of the disclosed EGFR RD-binding antibody.

Also disclosed are methods for predicting prognosis of a neoplasticdisease (such as lung cancer, colorectal cancer, head and neck cancer,gastric cancer, or glioblastoma), including detecting in a biologicalsample from a patient having a neoplastic disease the specific bindingof a disclosed EGFR RD-binding antibody to one or more EGFR-positiveneoplastic cells in the biological sample; wherein the specific bindingof the antibody in the one or more EGFR-positive neoplastic cellspredicts a poor prognosis of the neoplastic disease in the patient. Insome such method embodiments, the antibody specifically binds to atleast 10% (such as at least 15%, at least 20%, at least 30%, at least50%, or at least 75%) of the EGFR-positive neoplastic cells in thebiological sample. In some method embodiments, a poor prognosis is lessthan 5-year survival (such as less than 1-year survival or less than2-year survival) of the patient after initial diagnosis of theneoplastic disease.

Other prognostic method embodiments involve detecting in a biologicalsample from a patient having a neoplastic disease (such as lung cancer,colorectal cancer, head and neck cancer, gastric cancer, orglioblastoma) the specific binding of a disclosed EGFR RD-bindingantibody to one or more EGFR-positive neoplastic cells in the biologicalsample; wherein substantially no specific binding of the antibody in theone or more EGFR-positive neoplastic cells predicts a good prognosis ofthe neoplastic disease in the patient. In some method embodiments, agood prognosis is greater than 2-year survival (such as greater than3-year survival, greater than 5-year survival, or greater than 7-yearsurvival) of the patient after initial diagnosis of the neoplasticdisease.

Immunostaining methods also are disclosed. Such methods involvecontacting a biological sample, comprising one or more cells, with adisclosed EGFR RD-binding antibody, and detecting the specific bindingof the antibody to an antigen (e.g., EGFR) in the one or more cells.

Other disclosed methods involve detecting a direct interaction betweenEGFR and an EGFR regulatory protein (such as a SOCS protein, e.g., SOCS1or SOCS3), by contacting a biological sample, comprising one or moreEGFR-positive cells, with a disclosed EGFR RD-binding antibody, anddetecting the specific binding of the antibody to the one or moreEGFR-positive cells, wherein the specific binding of the antibody to theone or more EGFR-positive cells detects that EGFR is not significantlyinteracting with an EGFR regulatory protein, wherein an interactionbetween EGFR and the EGFR regulatory protein masks the epitope of theantibody.

Other methods of detecting a direct interaction between EGFR and an EGFRregulatory protein (such as a SOCS protein, e.g., SOCS1 or SOCS3) aredisclosed. Such methods involve contacting a biological sample,comprising one or more EGFR-positive cells, with a disclosed EGFRRD-binding antibody, and detecting the specific binding of the antibodyto the one or more EGFR-positive cells, wherein substantially nospecific binding of the antibody to the one or more EGFR-positive cellsdetects that EGFR is interacting with an EGFR regulatory protein,wherein an interaction between EGFR and the EGFR regulatory proteinmasks the epitope of the antibody.

In any disclosed method embodiment involving a biological sample, suchbiological sample can be (but is not necessarily) mounted on amicroscope slide, is a tissue section (such as a formalin-fixed andparaffin-embedded tissue section), and/or is a neoplastic tissue (suchas, a lung cancer, colorectal cancer, head and neck cancer, gastriccancer, or glioblastoma).

II. Abbreviations and Terms

EGFR epidermal growth factor receptor (e.g., OMIM Accession No. 131550)IHC immunohistochemistry NSCLC non-small cell lung cancer RDP EGFRregulatory domain peptide RD EGFR regulatory domain SH2 domain Srchomology 2 domain SOCS protein suppressor of cytokine signaling protein(e.g., OMIM Accession Nos. 604176 or 603597) STAT signal transducer andactivator of transcription

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in cell and molecularbiology may be found in Benjamin Lewin, Genes V, published by OxfordUniversity Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of various embodiments of a disclosedinvention, the following explanations of specific terms are provided:

Antigen-binding molecule: A molecule that specifically binds to anepitope in a target molecule (e.g., an antigen, such as a protein ornucleic acid molecule). Exemplary antigen-binding molecules are providedelsewhere in this disclosure.

Species of antigen-binding molecules described herein include, withoutlimitation, interface-specific binding molecules, RD-binding molecules,and control antigen-binding molecules. These species of antigen-bindingmolecules are characterized by the nature of the target molecule and/orthe location in the target molecule of the epitope to which the speciesspecifically binds as more particularly defined elsewhere in thisdisclosure.

Epitope: A site on a target molecule (e.g., an antigen, such as aprotein or nucleic acid molecule) to which an antigen-binding molecule(e.g., an antibody, antibody fragment, scaffold protein containingantibody binding regions, or aptamer) binds. Epitopes can be formed bothfrom contiguous or juxtaposed noncontiguous residues (e.g., amino acidsor nucleotides) of the target molecule. Epitopes formed from contiguousresidues (e.g., amino acids or nucleotides) typically are retained onexposure to denaturing solvents whereas epitopes formed by tertiaryfolding typically are lost on treatment with denaturing solvents. Anepitope typically includes at least 3, and more usually, at least 5 or8-10 residues (e.g., amino acids or nucleotides). Typically, an epitopealso is less than 20 residues (e.g., amino acids or nucleotides) inlength, such as less than 15 residues or less than 12 residues.

Immunogen: A molecule (also called an antigen) capable of provoking animmune response (e.g., the production of antibodies) when introducedinto an animal with a functioning immune system. Exemplary immunogensincluding, for instance, proteins (or protein fragments),polysaccharides, and small molecules (haptens) or peptides coupled to acarrier molecule (e.g., a protein such as bovine serum albumin (“BSA”),keyhole limpet hemocyanin (“KLH”) or polylysine). An “immunogenicfragment” is a portion of a polypeptide or other immunogen that iscapable of provoking an immune response either by itself or whenconjugated to a carrier molecule. Immunogens and immunogenic fragmentsinclude one or more epitopes within their sequences.

Isolated: An “isolated” biological component (e.g., a nucleic acidmolecule, chemical compound, protein or organelle) has beensubstantially separated or purified away from other biologicalcomponents (e.g., nucleic acid molecules, chemical compounds, proteinsor organelles) with which the component is commingled (e.g., in the cellof an organism or in a plant cell extract). Nucleic acids, proteins andchemical compounds that have been “isolated” include nucleic acids,proteins and chemical compounds purified by standard purificationmethods. The term “isolated” also embraces nucleic acids and proteinsprepared by recombinant expression in a host cell as well as chemicallysynthesized nucleic acids and chemical compounds.

Peptide: Two or more amino acids joined by a peptide bond. Typically, apeptide consists of fewer than fifty amino acids; for example,consisting of approximately 7 to approximately 40 amino acids,consisting of approximately 7 to approximately 30 amino acids,consisting of approximately 7 to approximately 20 amino acids.

Specific binding (or obvious derivations of such phrase, such asspecifically binds, specific for, etc.) refers to the particularinteraction between one binding partner (such as an EGFR RD-bindingmolecule) and another binding partner (such as a target of an EGFRRD-binding molecule). Such interaction is mediated by one or, typically,more noncovalent bonds between the binding partners (or, often, betweena specific region or portion of each binding partner). In contrast tonon-specific binding sites, specific binding sites are saturable.Accordingly, one exemplary way to characterize specific binding is by aspecific binding curve. A specific binding curve shows, for example, theamount of one binding partner (the first binding partner) bound to afixed amount of the other binding partner as a function of the firstbinding partner concentration. As the first binding partnerconcentration increases under these conditions, the amount of the firstbinding partner bound will saturate. In another contrast to non-specificbinding sites, specific binding partners involved in a directassociation with each other (e.g., a protein-protein interaction) can becompetitively removed (or displaced) from such association (e.g.,protein complex) by excess amounts of either specific binding partner.Such competition assays (or displacement assays) are very well known inthe art.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which a disclosed invention belongs. The singularterms “a,” “an,” and “the” include plural referents unless contextclearly indicates otherwise. Similarly, the word “or” is intended toinclude “and” unless the context clearly indicates otherwise.“Comprising” means “including.” Hence “comprising A or B” means“including A” or “including B” or “including A and B.

Suitable methods and materials for the practice and/or testing ofembodiments of a disclosed invention are described below. Such methodsand materials are illustrative only and are not intended to be limiting.Other methods and materials similar or equivalent to those describedherein can be used. For example, conventional methods well known in theart to which a disclosed invention pertains are described in variousgeneral and more specific references, including, for example, Sambrooket al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold SpringHarbor Laboratory Press, 1989; Sambrook et al., Molecular Cloning: ALaboratory Manual, 3d ed., Cold Spring Harbor Press, 2001; Ausubel etal., Current Protocols in Molecular Biology, Greene PublishingAssociates, 1992 (and Supplements to 2000); Ausubel et al., ShortProtocols in Molecular Biology: A Compendium of Methods from CurrentProtocols in Molecular Biology, 4th ed., Wiley & Sons, 1999; Harlow andLane, Antibodies: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, 1990; and Harlow and Lane, Using Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory Press, 1999.

All sequences associated with the GenBank Accession Nos. mentionedherein are incorporated by reference in their entirety as were presenton Jul. 13, 2007, to the extent permissible by applicable rules and/orlaw.

III. Methods of Determining Molecular Interactions in Fixed BiologicalSamples

This disclosure provides, among other things, methods for identifyingprotein-protein (or protein-nucleic acid) interactions in biologicalsamples (e.g., isolated cells or tissues) that have been mounted on asolid surface (e.g., a microscope slide) and treated (e.g.,formalin-fixed and paraffin-embedded (“FFPE”)) to substantially maintainthe positions of components (e.g., proteins, RNAs and/or DNA) within thesample relative to one another.

Molecular interactions (e.g., protein-protein interactions) previouslyhave been studied in solution and using in vivo techniques, such asco-immunoprecipitation assays (where a protein of interest is capturedwith an antibody and any interaction partners bound to the protein aresubsequently identified by Western blot); pull-down assays (which aresimilar to co-immunoprecipitation assays, but use some ligand other thanan antibody to capture the protein complex); label transfer (where aknown protein is tagged with a detectable label and the label is thenpassed to an interacting protein); in vivo crosslinking of proteincomplexes (where cells are grown under conditions that causephotoreactive diazirine amino acid analogs to be incorporated intocellular proteins, which diazirines can be activated and bind tointeracting proteins); the yeast two-hybrid screen (which investigatesthe interaction between artificial fusion proteins inside the nucleus ofyeast); and dual polarisation interferometry (“DPI”) (which providesreal-time, high-resolution measurements of molecular size, density andmass). Each of the foregoing methods requires means to isolate (whetherphysically, chemically or otherwise) the components having a specificinteraction with one another from other non-interacting components.

Non-specific crosslinking reactions (such as, chemical crosslinking)also may be useful to examine protein-protein interactions in settingswhere non-specific interactions between reaction components can becontrolled. However, biological samples (e.g., isolated cells ortissues) mounted on a solid surface (e.g., microscope slides or supportmembranes) do not offer such a setting. Under those conditions,non-specific crosslinkers bond together (permanently orsemi-permanently) any components in the sample that in proximity of eachother whether or not such components interact under biologicalconditions.

Rather than view non-specific crosslinking as a hindrance to examiningprotein-protein interactions, the present discovery actually exploitsthe non-specific crosslinking of biological components within a fixedbiological sample (e.g., FFPE cells or FFPE tissues). Such crosslinkingsubstantially ensures that the structural relationship betweeninteracting components in the sample (e.g., protein-protein orprotein-nucleic acid) is permanently or semi-permanently maintained;thereby, masking some or all residues that form the interface betweenthe components. For example, any epitope present in the interface wouldnot be available to a cognate antigen-binding protein (e.g., antibody)following fixation of the sample. Accordingly, the accessibility (ornot) of the residues within the interface to binding proteins (e.g.,antibodies) specific for such residues can be used to determine whetheror not the particular components were interacting in the biologicalsample at the time it was fixed.

Some disclosed methods involve identifying at least two biologicalcomponents (e.g., two proteins or a protein and a nucleic acid sequence)that together form a direct interaction, determining the residues (e.g.,amino acids or nucleotides) involved in the interface between the atleast two components, identifying or making at least one antigen-bindingmolecule (such as a monoclonal antibody or fragment thereof) specificfor some or all of the residues involved in the interface between the atleast two components, detecting in a fixed biological sample (such asFFPE tissue sections or fixed cell samples) the binding (or absence ofbinding) of the at least one interface-specific antigen-bindingmolecule. In some methods, the interacting components and the particularresidues (or regions) involved in the interface between the at least twocomponents are known; hence, identifying such components and the natureof their interface would be optional steps of the disclosed method.

Because fixation of the interface(s) between the at least twointeracting components (e.g., proteins or protein and nucleic acid)leads to the exclusion of interface-specific antigen-binding moleculesfrom binding residues in the interface(s), some methods will involve anegative result (i.e., no binding). In some such methods, it can beadvantageous to further detect the presence of one or more (e.g., one ortwo) components of the interaction complex; thus, showing that thefailure of the at least one interface-specific antigen-bindingmolecule(s) to bind its target(s) is not due to absence of one or moreof the components involved in the making of the interface(s) but ratheris due to the masking of the target(s).

Biological samples useful in a disclosed method are isolated and includeany cell preparation or tissue preparation that can be fixed and mounton a solid surface. Exemplary samples include, without limitation, bloodsmears, cytocentrifuge preparations, cytology smears, core biopsies,fine-needle aspirates, and/or tissue sections (e.g., cryostat tissuesections and/or paraffin-embedded tissue sections). Exemplary biologicalsamples may be isolated from normal cells or tissues, or from neoplasticcells or tissues. Neoplasia is a biological condition in which one ormore cells have undergone characteristic anaplasia with loss ofdifferentiation, increased rate of growth, invasion of surroundingtissue, and which cells may be capable of metastasis. Exemplaryneoplastic cells or tissues may be isolated from solid tumors, includingbreast carcinomas (e.g. lobular and duct carcinomas), sarcomas,carcinomas of the lung (e.g., non-small cell carcinoma, large cellcarcinoma, squamous carcinoma, and adenocarcinoma), mesothelioma of thelung, colorectal adenocarcinoma, stomach carcinoma, prostaticadenocarcinoma, ovarian carcinoma (such as serous cystadenocarcinoma andmucinous cystadenocarcinoma), ovarian germ cell tumors, testicularcarcinomas and germ cell tumors, pancreatic adenocarcinoma, biliaryadenocarcinoma, hepatocellular carcinoma, bladder carcinoma (including,for instance, transitional cell carcinoma, adenocarcinoma, and squamouscarcinoma), renal cell adenocarcinoma, endometrial carcinomas(including, e.g., adenocarcinomas and mixed Mullerian tumors(carcinosarcomas)), carcinomas of the endocervix, ectocervix, and vagina(such as adenocarcinoma and squamous carcinoma of each of same), tumorsof the skin (e.g., squamous cell carcinoma, basal cell carcinoma,melanoma, and skin appendage tumors), esophageal carcinoma, carcinomasof the nasopharynx and oropharynx (including squamous carcinoma andadenocarcinomas of same), salivary gland carcinomas, brain and centralnervous system tumors (including, for example, tumors of glial,neuronal, and meningeal origin), tumors of peripheral nerve, soft tissuesarcomas and sarcomas of bone and cartilage.

A solid support useful in a disclosed method need only bear thebiological sample and, optionally, but advantageously, permit theconvenient detection of components (e.g., proteins and/or nucleic acidsequences) in the sample. Exemplary supports include microscope slides(e.g., glass microscope slides or plastic microscope slides), coverslips(e.g., glass coverslips or plastic coverslips), tissue culture dishes,multi-well plates, membranes (e.g., nitrocellulose or polyvinylidenefluoride (PVDF)) or BIACORE™ chips.

Fixatives for mounted cell and tissue preparations are well known in theart and include, without limitation, 95% alcoholic Bouin's fixative; 95%alcohol fixative; B5 fixative, Bouin's fixative, formalin fixative,Karnovsky's fixative (glutaraldehyde), Hartman's fixative, Hollande'sfixative, Orth's solution (dichromate fixative), and Zenker's fixative(see, e.g., Carson, Histotechology: A Self-Instructional Text, Chicago:ASCP Press, 1997).

Biological components (e.g., proteins and/or nucleic acid sequences)that form direct interactions (such as protein-protein interactions) areknown to those of ordinary skill in the art. Various exemplaryprotein-protein interactions can be identified in one or more of thefollowing publicly available databases: AllFuse (European BioinformaticsInstitute), Alanine Scanning Energetics DataBase (ASEdb; HarvardUniversity), Binding Interface Database (BID; A & M University Texas);The General Repository for Interaction Datasets (BioGRID; SamuelLunenfeld Research Institute); Biomolecular Object Network Databank(BOND; Thomson Corp.); Database of Interacting Proteins (DIP; UCLA);Genomic Knowledge Database (RIKEN, Institute of Physical and ChemicalResearch); HIV-1/Human Protein Interaction Database (NCBI); HumanProtein Intercation Database (UPID; Inha University); Human ProteinReference Database (Johns Hopkins University and The Institute ofBioinformatics, India); Inter-Chain Beta-Sheets database (ICBS;University of California); Kinetic Data of Bio-molecular Interactions(KDBI; National University of Singapore); Biomolecular Relations inInformation Transmission and Expression (KEGG BRITE; Kyoto University);Molecular INTeractions database (MINT; CBM, Rome); MammalianProtein-Protein Interaction database (MPPI; MIPS); PDZBase (WeillMedical College of Cornell University); POINT (National Health ResearchInstitutes & National Taiwan University); PRotein Interactions andMolecular Information databasE (PRIME; Human Genome Center, Universityof Tokyo); Protein Interaction Database (Protein Lounge); SNAPPIView(University of Dundee).

Some of the foregoing databases further identify the residues or regionsof the applicable proteins involved in the protein-protein interface.Alternatively, residues or regions involve in a protein-proteininteraction can be determined using any technique known to theordinarily skilled artisan; for example, peptide competition studies(where a peptide having a sequence corresponding to residues believed tobe involved in a protein-protein interface is used to competitivelyinhibit the protein-protein interaction; successful inhibition by thepeptide of the interaction indicates that the subject sequence likely isinvolved in the protein-protein interaction), or mutational analysis ofone or both components of the protein-protein interaction.

Once a region or residues of an interface between directly interactingproteins (or a protein and nucleic acid sequence) is known ordetermined, a binding molecule that specifically recognizes theinterface region or an epitope consisting of interface residues (i.e.,an interface-specific binding molecule) can be obtained from acommercially available source or prepared using techniques common in theart. For example, methods of preparing antibodies, antibody fragments,aptamers and other antigen-binding molecules are described in detailelsewhere in this disclosure.

Particular method embodiments involve detecting in a fixed biologicalsample a protein complex including (or consisting of) EGFR and anEGFR-interacting protein (e.g., a regulatory protein, such as a SOCSprotein like SOCS1 or SOCS3). EGFR is known to form protein-proteininteractions in vivo and in vitro with numerous other proteins. Somesuch interactions are listed in Table 1.

TABLE 1 Exemplary EGFR Interaction Partners PARTNER 1 PARTNER 2 SYSTEMSOURCE PUBMED ID AREG EGFR In vivo Wong L et al. 10085134 CD44 EGFR Invivo Tsatas D et al. 12093135 EGFR GRB2 In vivo Lowenstein EJ et al.1322798 EGFR GRB2 In vivo Okutani T et al. 7527043 EGFR GRB2 In vitroLowenstein EJ et al. 1322798 EGFR GRB2 In vitro Okutani T et al. 7527043EGFR CTNNB1 In vivo Takahashi K et al. 9233779 EGFR CDC25A In vivo WangZ et al. 11912208 EGFR CDC25A In vitro Wang Z et al. 11912208 DCN EGFRIn vivo Santra M et al. 12105206 DCN EGFR In vivo Iozzo RV et al.9988678 DCN EGFR In vitro Santra M et al. 12105206 DCN EGFR In vitroIozzo RV et al. 9988678 DCN EGFR Two-hybrid Santra M et al. 12105206 DCNEGFR Two-hybrid Iozzo RV et al. 9988678 EGFR HBEGF In vivo Shin SY etal. 12725245 EGF EGFR In vitro Stortelers C et al. 12093292 EGFR CAV1 Invivo Couet J et al. 9374534 EGFR CAV1 In vitro Couet J et al. 9374534EGFR PRKACA In vivo Tortora G et al. 9050991 EGFR ERBB3 In vivo MarquesMM et al. 10527633 EGFR SHC1 In vitro Sakaguchi K et al. 9544989 ITGA5EGFR In vitro Kuwada SK et al. 10888683 ITGA5 EGFR In vivo Kuwada SK etal. 10888683 EGFR ZNF259 In vivo Moores SL et al. 10938113 EGFR RASA1 Invitro Serth J et al. 1633149 PTK2 EGFR In vivo Sieg DJ et al. 10806474PTK2 EGFR In vitro Sieg DJ et al. 10806474 PLSCR1 EGFR In vivo Sun J etal. 12009895 PLSCR1 EGFR In vivo Nanjundan M et al. 12871937 PLSCR1 EGFRIn vitro Sun J et al. 12009895 PLSCR1 EGFR In vitro Nanjundan M et al.12871937 GRB14 EGFR In vivo Daly RJ et al. 8647858 GRB14 EGFR In vitroDaly RJ et al. 8647858 EGFR DOK2 In vitro Jones N et al. 10508618 KRT7EGFR In vivo Blagoev B et al. 12577067 KRT7 EGFR In vitro Blagoev B etal. 12577067 TGFA EGFR In vitro Garrett TP et al. 12297049 EGFR XRCC6 Invivo Bandyopadhyay D et al. 9430697 EGFR PLD2 In vitro Slaaby R et al.9837959 EGFR PLD2 In vivo Slaaby R et al. 9837959 CD82 EGFR In vivoOdintsova E et al. 10985391 CD82 EGFR In vitro Odintsova E et al.10985391 SNX6 EGFR In vivo Parks WT et al. 11279102 SNX6 EGFR In vitroParks WT et al. 11279102 EGFR PTK2B In vivo Keely SJ et al. 10777553GRB10 EGFR In vivo Frantz JD et al. 9006901 GRB10 EGFR In vivo He W etal. 9506989 GRB10 EGFR In vitro Frantz JD et al. 9006901 GRB10 EGFR Invitro He W et al. 9506989 PRKCA EGFR In vivo Gauthier ML et al. 12878187PRKCA EGFR In vitro Gauthier ML et al. 12878187 EGFR STAT1 In vivo Xia Let al. 12070153 EGFR STAT1 In vitro Xia L et al. 12070153 KRT18 EGFR Invivo Blagoev B et al. 12577067 KRT18 EGFR In vitro Blagoev B et al.12577067 EGFR SOCS3 In vivo Xia L et al. 12070153 EGFR SOCS3 In vitroXia L et al. 12070153 EGFR MIG-6 In vivo Hackel PO et al. 11843178 EGFRMIG-6 Two-hybrid Hackel PO et al. 11843178 EGFR SOCS1 In vivo Xia L etal. 12070153 PLEC1 EGFR In vivo Blagoev B et al. 12577067 PLEC1 EGFR Invitro Blagoev B et al. 12577067 EGFR SNX4 In vivo Haft CR et al. 9819414EGFR PRKAR1A In vivo Tortora G et al. 9050991 AMH EGFR In vitro MaggardMA et al. 8596488 EGFR MAP4K1 In vivo Anafi M et al. 9346925 PLCG1 EGFRIn vivo Bedrin MS et al. 9207933 SNX1 EGFR In vivo Haft CR et al.9819414 SNX1 EGFR In vivo Kurten RC et al. 8638121 SNX1 EGFR Two-hybridHaft CR et al. 9819414 SNX1 EGFR Two-hybrid Kurten RC et al. 8638121EGFR STAT5B In vivo Runge DM et al. 10558875 DEGS1 EGFR In vivo CadenaDL et al. 9188692 DEGS1 EGFR In vitro Cadena DL et al. 9188692 DEGS1EGFR Two-hybrid Cadena DL et al. 9188692 EGFR PIK3C2B In vitro Wheeler Met al. 11533253 KRT17 EGFR In vivo Blagoev B et al. 12577067 KRT17 EGFRIn vitro Blagoev B et al. 12577067 RGS16 EGFR In vivo Derrien A et al.11602604 HD EGFR In vivo Liu YF et al. 9079622 PITPNA EGFR In vivoKauffmann-Zeh A et al. 7761838 PITPNA EGFR In vitro Kauffmann-Zeh A etal. 7761838 EGFR CEBPB In vivo Harmon AW et al. 12095417 EGFR CEBPB Invitro Harmon AW et al. 12095417 MUC1 EGFR In vivo Li Y et al. 11483589MUC1 EGFR In vivo Schroeder JA et al. 11278868 NCK2 EGFR In vivo Chen Met al. 9737977 NCK2 EGFR In vivo Li W et al. 1333047 EGFR PTK6 In vivoKamalati T et al. 8940083 EGFR CAV3 In vivo Couet J et al. 9374534 EGFRCAV3 In vitro Couet J et al. 9374534 EGFR CRK In vivo Hashimoto Y et al.9642287 EGFR CRK In vitro Hashimoto Y et al. 9642287 EGFR GRB7 In vivoTanaka S et al. 9710451 EGFR GRB7 In vitro Tanaka S et al. 9710451 EGFRSNRPD2 In vivo Blagoev B et al. 12577067 EGFR SNRPD2 In vitro Blagoev Bet al. 12577067 EGFR SRC In vitro Sato K et al. 10971656 CBLB EGFR Invivo Ettenberg SA et al. 10086340 CBLB EGFR In vitro Ettenberg SA et al.10086340 EGFR CBLC In vivo Keane MM et al. 10362357 EGFR CBLC In vitroKeane MM et al. 10362357 EGFR SHC1 In vitro Blagoev B et al. 12577067EGFR PTPN6 In vivo Keilhack H et al. 9733788 EGFR PTPN6 In vitroKeilhack H et al. 9733788 EPS8 EGFR In vivo Castagnino P et al. 7532293EPS8 EGFR In vivo Di Fiore PP et al. 12127568 SNX2 EGFR In vivo Haft CRet al. 9819414 SNX2 EGFR In vitro Haft CR et al. 9819414 TNC EGFR Invivo Swindle CS et al. 11470832 TNC EGFR In vitro Swindle CS et al.11470832 EGFR CAMLG In vivo Tran DD et al. 12919676 EPPK1 EGFR In vivoBlagoev B et al. 12577067 EPPK1 EGFR In vitro Blagoev B et al. 12577067EGFR NCK1 In vitro Tang J et al. 9362449 KRT8 EGFR In vivo Blagoev B etal. 12577067 KRT8 EGFR In vitro Blagoev B et al. 12577067 EGFR TJP1 Invivo Kaihara T et al. 12708492 VAV1 EGFR In vivo Moores SL et al.10938113 EGFR STAT5A In vivo Olayioye MA et al. 10358079 EGFR GAB1 Invivo Kameda H et al. 11432805 EGFR TNK2 In vivo Manser E et al. 8497321EGFR TNK2 In vivo Satoh T et al. 8647288 EGFR VAV2 In vivo Pandey A etal. 10618391 EGFR VAV2 In vivo Moores SL et al. 10938113 EGFR VAV2 Invitro Pandey A et al. 10618391 EGFR VAV2 In vitro Moores SL et al.10938113 SOS1 EGFR In vivo Qian X et al. 9447973 EGFR MAP3K14 In vivoHabib AA et al. 11116146 EGFR ESR1 In vitro Marquez DC et al. 11887937EGFR ERBB2 In vivo Brockhoff G et al. 11500850 EGFR ERBB2 In vitroBrockhoff G et al. 11500850 SH2D3A EGFR In vivo Lu Y et al. 10187783CDH1 EGFR In vivo Pece S et al. 10969083 EGFR PTPN1 In vivo Zhang ZY etal. 8621392 EGFR PTPN1 In vivo Jia Z et al. 7540771 EGFR PTPN1 In vivoSarmiento M et al. 10889023 EGFR PTPN1 In vivo Li S et al. 12573287 EGFRPTPN1 In vitro Zhang ZY et al. 8621392 EGFR PTPN1 In vitro Jia Z et al.7540771 EGFR PTPN1 In vitro Sarmiento M et al. 10889023 EGFR PTPN1 Invitro Li S et al. 12573287 VAV3 EGFR In vivo Zeng L et al. 11094073INPPL1 EGFR In vitro Pesesse X et al. 11349134 INPPL1 EGFR In vivoPesesse X et al. 11349134 EPS15 EGFR In vivo van Delft S et al. 9049247EGFR ARF4 In vitro Kim SW et al. 12446727 EGFR ARF4 In vivo Kim SW etal. 12446727 EGFR ARF4 Two-hybrid Kim SW et al. 12446727 EGFR GNAI2 Invivo Zhang BH et al. 11286993 EGFR PDGFRB In vivo Habib AA et al.9506992 SHC3 EGFR In vivo Nakamura T et al. 9507002 EGFR BTC In vivoMixan B et al. 9528863 EGFR SOS2 In vivo Qian X et al. 10675333 NRG1EGFR In vitro Pinkas-Kramarski R et al. 8702572 EGFR CASP1 In vivo BaeSS et al. 11226410 EGFR CASP1 In vitro Bae SS et al. 11226410 EGFR EREGIn vivo Komurasaki T et al. 9419975 EGFR PTPN11 In vitro Tomic S et al.7673163 EGFR PTPN11 In vivo Tomic S et al. 7673163 PTPRJ EGFR In vivoJallal B et al. 9115287 EGFR RIPK1 In vivo Habib AA et al. 11116146 FEREGFR In vivo Kim L et al. 7623846

Protein-protein interactions typically influence the activity of one orboth interacting partners. For example, a protein-protein interactionmay result in the negative regulation (e.g., inhibition) of one or bothpartners, or may result in the positive regulation (e.g. activation) ofone or both partners. Other functional outcomes also are possible.Exemplary negative regulators of EGFR that form protein-proteininteractions with EGFR include, for instance, SOCS1, SOCS3, SOCS5, andC-CBL. Exemplary positive regulators of EGFR that form protein-proteininteractions with EGFR include, for instance, STAT1, STAT5B, GRB7, BER2,and MUC1.

Methods useful for detection of a protein-protein interaction using aninterface-specific binding molecule (such as an antibody, antibodyfragment, recombinant antibody, scaffold polypeptide with antibodybinding sites, and/or an aptamer) are well known in the art. In someexamples, a fixed biological sample is contacted with aninterface-specific binding molecule under conditions that permit (orwould permit if it was accessible) binding of the interface-specificbinding molecule to its epitope in the interface between the interactingproteins. Optionally, a control reaction is performed (e.g.,simultaneous with, prior to, or following) to ensure that the conditionsare suitable for the detection reaction to occur. For example, thebiological sample (or a serial section or a parallel-prepared cellsample) also may be contacted with a control antigen-binding molecule(such as, an antibody, antibody fragment, recombinant antibody, scaffoldpolypeptide with antibody binding sites, and/or an aptamer). The controlantigen-binding molecule specifically binds to a non-interactingcomponent of the sample (i) that is not involved in the protein-proteincomplex of interest and (ii) the epitope of which is known to be presentand detectable in the sample under the particular detection conditions.

In exemplary methods, an interface-specific binding molecule and anoptional control antigen-binding molecule are antibodies (e.g.,monoclonal antibodies) or antibody fragments. Detection of suchantibodies or antibody fragments is performed by immunostaining, such asillustrated in FIG. 1, which is a standard technique in the art.Detection may by direct or indirect. With direct detection, primaryantibodies (i.e., the antibodies that specifically bind to biologicalcomponent(s) of interest in the sample) are directly labeled, forinstance, with a detectable moiety or with an enzyme that catalyzes areaction leading to a detectable product. With indirect detection, oneor more secondary reagents (such as secondary, tertiary, etc.antibodies) are used to detect the primary antibody (and, as applicable,secondary or subsequent antibodies) and the last of such reagents isdetectable, for instance by labeling with detectable moiety or with anenzyme that catalyzes a reaction leading to a detectable product.Representative immunostaining procedures are provided in the Examples.

Some disclosed methods involve dual detection of an external domainepitope present on EGFR and an internal domain (or regulatory domain)epitope of EGFR. The EGFR regulatory domain has multiple binding sitesfor regulatory proteins (see, e.g., FIG. 2). Accordingly, epitopes inthe EGFR regulatory domain may not be accessible in fixed biologicalsamples where, at the time of fixation, EGFR was involved in aprotein-protein interaction with one or more of its regulatory proteins(such as a SOCS protein, e.g., SOCS1 or SOCS3). In comparison, the EGFRexternal domain functions primary as a ligand-binding domain, and,typically, it is not masked from antibodies specific for external domainepitopes. Accordingly, in some methods, an antigen-binding moleculespecific for the EGFR external domain (anti-EGFR external domainantibody or fragment thereof) can serve as a control for anantigen-binding molecule specific for a (potentially masked) epitope inan EGFR-regulatory protein interface. One caveat of interest is thatEGFR may “shed” its external domain or may be mutant and lack itsexternal domain (Pedersen et al., Ann. Oncol., 6:745, 2001). Under thosecircumstances, an antigen-binding molecule specific for the EGFRexternal domain (e.g., anti-EGFR external domain antibody or fragmentthereof) would have no target to bind and, therefore, would not bedetected. This circumstance (and useful information that can be gleanedfrom such circumstance) is discussed in detail elsewhere in thisdisclosure.

Some of the foregoing method embodiments and other method embodiments inthis disclosure involve substantially no specific binding of aRD-binding molecule (such as a monoclonal antibody) to its epitope(e.g., which is located in the protein-protein interface between EGFRand it regulatory molecule(s)). Substantially no binding can bedetermined by any method available to those of ordinary skill in theart. For example, substantially no binding of a RD-binding molecule maybe relative to the binding of the same RD-binding molecule undersubstantially the same conditions in another sample in which the epitopeof the RD-binding molecule is known to be accessible. In anotherexample, substantially no binding of a RD-binding molecule may mean thatthe detection means (e.g., detectable label or calorimetric reagent)used to visualize the specific binding of the RD-binding molecule cannot be seen under ordinary circumstances for such detection, e.g., undera light or fluorescence microscope with 4×, 10×, or 40× magnification.In still another example, substantially no binding of a RD-bindingmolecule means that the RD-binding molecule has less than about 25%(such as less than about 20%, less than about 15%, less than about 10%,less than about 5%, or less than about 1%) its binding under controlcircumstances (e.g., in a tissue or cell sample where its epitope isknown to be accessible).

IV. Predictive Methods

The discovery herein of methods to detect EGFR molecular interactions infixed biological samples opens the way to predicting EGFR status andimportant corollaries in such samples or in subjects from which suchsamples are collected; for example, in neoplastic tissues and/or cellswhere EGFR overexpression is believed to play an important role intumorigenesis (e.g., Arnold et al., Oncologist, 6:602, 2006) and/or incancer patients. The disclosed predictive methods are applicable to anytype of cancer or to a subject with any type of cancer, for instanceEGFR-expressing (or -overexpressing) cancers. Exemplary neoplasms usefulin all disclosed methods (including predictive methods) are describedelsewhere in this disclosure (e.g., Section III and the Examples).Particular predictive method embodiments involve lung cancer (e.g.,non-small cell lung cancer), ovarian cancer, colorectal cancer, livercancer, head and neck, prostate, and/or glioblastoma and/or subjectshaving any of such cancers.

A. Predicting Aggressiveness of EGFR-Positive Neoplasms

Detection of a direct (e.g., protein-protein) interaction between EGFRand a negative regulator of EGFR function (e.g., a SOCS protein, such asSOCS1 or SOCS3, or SOCS5) predicts inhibition of EGFR function in thatbiological sample. Inhibition of EGFR function has importantconsequences in many cells and tissues. For example, in neoplastic cellsand tissues where EGFR overexpression is believed to play a role intumorigenesis (e.g., Arnold et al., Oncologist, 6:602, 2006), detectionof a direct interaction between EGFR and a negative regulator of EGFRfunction (e.g., a SOCS protein, such as SOCS1 or SOCS3) further predictsthat a neoplasm may be less aggressive (e.g., less rapidly growing,and/or less likely to metastasize). A better prognosis (independent oftherapy) for a subject with such a neoplasm also may be predicted.

On the other hand, detection of a direct interaction between EGFR and apositive regulator of EGFR function (e.g., STAT1, STAT5B, GRB7, BER2,and/or MUC1) predicts activation of EGFR function in that biologicalsample (e.g., neoplastic tissue or cells). For the opposite of reasonsdiscussed above, a worse prognosis (independent of therapy) for asubject with such a neoplasm also may be predicted.

A less-aggressive tumor can be characterized by any parameters known inthe art, including, for instance, decreased growth rate (e.g., increasedrate of apoptosis and/or decreased rate of cell division), decreasedrate of metastasis, and/or increased sensitivity to chemotherapy.

Prognosis for a subject can be characterized by any parameter known inthe art, including, for instance, actual survival after initialdiagnosis (such as 6-month survival, 1-year survival, 2-year survival,or 5-year survival), and/or actual survival relative to the averagesurvival for similarly situated patients. A better prognosis entails,e.g., survival of a patient for more than 1 year after initial diagnosis(such as more than 2 years or more than 5 years), or survival of apatient for more than 6 months longer (e.g., more than 1 year longer,more than 2 years longer, more than 5 years longer) than the averagesurvival for similarly situated. A worse prognosis entails, e.g.,survival of a patient for less than 5 years after initial diagnosis(such as less than 2 years or less than 1 years), or survival of patientless than the average survival for similarly situated patients (such as,about 3 months less than average survive, about 6 months less thanaverage survive, or about 1 year less than average survival).

Exemplary prognoses based on detecting an interaction (or lack ofinteraction) between EGFR and, e.g., a negative regulator that binds theEGFR regulatory domain (such as a SOCS protein like SOCS1 and/or SOCS3)are shown schematically in FIG. 3.

B. Predicting Responsiveness of a Cancer Patient to EGFR-InhibitorTherapy

Methods of detecting an interaction (or lack of interaction) betweenEGFR and, e.g., its negative regulator(s) (such as a SOCS protein likeSOCS1 and/or SOCS3) enables a variety of predictions with respect to theoutcome of EGFR inhibitor therapy in a cancer patient. EGFR inhibitortherapies include at least two drug classes: EGFR antibody therapies(such as, cetuximab (Erbitux™), panitumumab (Vectibix™), IMC-11F8(Imclone), matuzumab (Merck_KGA)) and tyrosine kinase inhibitors(“TKIs”) (such as gefitinib (Iressa™), erlotinib (Tarceva™), lapatinibditosylate (GlaxoSmithKline), HKI-272 (Wyeth), AEE788 (Novartis),vandetanib (Zactima™; Astrazeneca), XL647 (Exelixis), BMS-599626(Bristol-Myers Squibb), BIBW 2992 (Boehringer Ingelheim)). EGFR antibodytherapies typically are directed to the EGFR external domain and blockbinding of an EGFR ligand (such as EGF) to the receptor; thereby,inhibiting EGFR activation. TKIs work by inhibiting the intracellularkinase domain of EGFR, which also inhibits EGFR activation.

Some method embodiments involve one or both of the foregoing classes ofEGFR inhibitors. Particular method embodiments involve predicting theresponse of cancer patients to cetuximab (Erbitux™), panitumumab(Vectibix™), gefitinib (Iressa™), or erlotinib (Tarceva™), or anycombination thereof (such as, cetuximab (Erbitux™) or panitumumab(Vectibix™), gefitinib (Iressa™) or erlotinib (Tarceva™), or cetuximab(Erbitux™), panitumumab (Vectibix™), gefitinib (Iressa™) or erlotinib(Tarceva™)).

Exemplary predictions based on detecting an interaction (or lack ofinteraction) between EGFR and, e.g., a negative regulator that binds theEGFR regulatory domain (such as a SOCS protein like SOCS1 and/or SOCS3)are provided in Table 2 and shown schematically in FIG. 3.

TABLE 2 Exemplary Predicted Therapeutic Response Predicted ID-BindingED-Binding Therapeutic Molecule Molecule EGFR Implication TherapeuticResponse 1 Positive Positive EGFR is present ED-based therapy SensitiveRDIM (e.g. SOCS3) (e.g., EGFR Ab) is absent ID-based therapy Sensitive(e.g., TKI) 2 Negative Positive EGFR is present ED-based therapyResistant RDIM (e.g. SOCS3) (e.g., EGFR Ab) is present ID-based therapyResistant (e.g., TKI) 3 Positive Negative Mutant/Cleaved ED-basedtherapy Resistant EGFR is present (e.g., EGFR Ab) RDIM (e.g. SOCS3)ID-based therapy Sensitive is absent (e.g., TKI) 4 Negative NegativeEGFR is absent ED-based therapy No Response (e.g., EGFR Ab) ID-basedtherapy No Response (e.g., TKI) ID = EGFR internal (or regulatory)domain; ED = EGFR extracellular domain; RDIM = regulatory domaininhibitory molecule; Ab = antibody (e.g., monoclonal or otherwiseengineered antibody)

In one method embodiment, an interaction between the internal regulatorydomain of EGFR and its negative regulator (e.g., a SOCS protein, such asSOCS1 and/or SOCS3) is detected (e.g., by masking of the epitope of aninterface-specific binding molecule (such as a monoclonal antibody,including clone 5B7 (see, e.g., Examples)). Optionally, butadvantageously, the presence of full-length (or substantiallyfull-length) EGFR also is detected using an antigen-binding molecule(e.g., monoclonal antibody, including clone 3C6) specific for the EGFRexternal domain. In this example, the interface-specific bindingmolecule (e.g., clone 5B7) is excluded from its binding site and,therefore, is not detected, while the external-domain antigen-bindingmolecule (e.g., clone 3C6) binds to its epitope and is detected. Thesecircumstances support a prediction that a therapy designed to inhibitEGFR function likely would not be effective or would be less effectivethan in the absence of the negative regulator. That is (solely forillustration purposes (and not to be limited by mechanism or implicationof a mechanism)): Providing an EGFR inhibitor to a subject in which EGFRfunction already was inhibited maybe analogous to applying the brakes ina car that is already at a stop.

In another method embodiment, an interaction between the internalregulatory domain of EGFR and its negative regulator (e.g., a SOCSprotein, such as SOCS1 and/or SOCS3) is lacking (e.g., as demonstratedby the binding to EGFR of an interface-specific binding molecule (suchas a monoclonal antibody, including clone 5B7 (see, e.g., Examples)) toits epitope, which would otherwise be masked by the EGFR-negativeregulator interaction). Optionally, but advantageously, the presence ofthe EGFR external domain (i.e., full-length or substantially full-lengthEGFR) also is detected using an antigen-binding molecule (e.g.,monoclonal antibody, including clone 3C6) specific for that domain. Inthis example, the interface-specific binding molecule (e.g., clone 5B7)specifically binds its epitope in the EGFR regulatory domain and,therefore, is detected, and the external-domain antigen-binding molecule(e.g., clone 3C6) also binds to its epitope and is detected. Thesecircumstances support a prediction that a therapy designed to inhibitEGFR function likely would be effective or would be more effective thanin the presence of the negative regulator.

In still another method embodiment, an interaction between the internalregulatory domain of EGFR and its negative regulator (e.g., a SOCSprotein, such as SOCS1 and/or SOCS3) is lacking (e.g., as demonstratedby the binding to EGFR of an interface-specific binding molecule (suchas a monoclonal antibody, including clone 5B7 (see, e.g., Examples)) toits epitope, which would otherwise be masked by the EGFR-negativeregulator interaction). Optionally, but advantageously, the presence orabsence of the EGFR external domain (i.e., full-length or substantiallyfull-length EGFR) also is detected using an antigen-binding molecule(e.g., monoclonal antibody, including clone 3C6) specific for thatdomain. In this example, the interface-specific binding molecule (e.g.,clone 5B7) specifically binds its epitope in the EGFR regulatory domainand, therefore, is detected; however, it also is determined that theEGFR external domain is lacking (e.g., a mutant or N-terminal truncatedEGFR) by failure to bind of an antigen-binding molecule specific forthat domain (e.g., monoclonal antibody, including clone 3C6). Thesecircumstances support a prediction that an antibody therapy designed toinhibit EGFR function by blocking ligand binding to the EGFR externaldomain likely would be not effective because such domain is lacking. Onthe other hand, these circumstances further support a prediction that aTKI therapy, which inhibits the tyrosine kinase activity localized inthe EGFR intracellular domain, likely would be effective or would bemore effective than in the presence of the negative regulator.

The response of a subject to EGFR inhibitor therapy can be measured byany relevant parameter known in the art. In some method embodiments, asubject response is cessation or slowing of tumor growth (as measured,for example, by tumor size), decrease in tumor cell proliferation,increase in tumor cell apoptosis, and/or decreased level of relevanttumor marker(s). In other method embodiments, a subject response is atleast a 50% slowing of tumor growth or tumor cell proliferation ascompared to pre-treatment growth (such at least a 40% slowing, at leasta 30% slowing, at least a 20% slowing, or at least a 10% slowing). Inother method embodiments, a subject response is at least a 50% increasein tumor cell apoptosis as compared to pre-treatment levels (such atleast a 40% increase, at least a 30% increase, at least a 20% increase,or at least a 10% increase).

V. EGFR Regulatory Domain Peptides

This disclosure concerns, among other things, the discovery of a19-amino acid region of EGFR that can be used, e.g., to interrogate thestructural and/or functional state of the receptor. This region has thesequence: LDNPDYQQDFFPKEAKPNG (SEQ ID NO: 2; “L2G Peptide”). It is foundin the C-terminal, intracellular (or cytoplasmic) domain of EGFR (forexemplary EGFR sequences, see, e.g., GENBANK™ Accession Nos.XP_(—)001156546.1; XP_(—)001156495.1; XP_(—)519102.2; XP_(—)001156439.1;BAD92679.1; AAS07524.1; AAX41033.1; NP_(—)113695.1; AAT52212.1;NP_(—)005219.2; and CAA25240.1, wherein the sequence present on Jul. 13,2007 is herein incorporated by reference).

The intracellular domain of EGFR, which corresponds to residues 669-1210of SEQ ID NO: 1, includes a kinase domain (residues 712-979 of SEQ IDNO: 1) and a regulatory domain (residues 980-1210). The EGFR regulatorydomain includes at least five tyrosine residues (Tyr1016, Tyr1092,Tyr1110, Tyr1172, and Tyr1197 of SEQ ID NO: 1), which are believed to beautophosphorylation sites (Chattopadhyay et al., J. Biol. Chem.,274:26091-7, 1999). Among all of the C-terminal tyrosine residues, thereare three YXXL/V (SEQ ID NO: 4) and four YXXP/D (SEQ ID NO: 5) motifs,which, for many transmembrane receptors, serve as the docking sites forSrc homology 2 (SH2) domain-containing proteins (Xia et al., J. Biol.Chem., 277(34):30716-23, 2002). As a class, SH2 domain-containingproteins are accepted phosphorylation-dependent regulators ofintracellular signal cascades.

The EGFR regulatory domain contains an inhibitory subdomain (Xia et al.,J. Biol. Chem., 277(34):30716-23, 2002), which corresponds to residues1138-1196 of SEQ ID NO: 1. The L2G Peptide sequence is contained withinthis inhibitory subdomain. The inhibitory subdomain is believed at leastto mediate a protein-protein interaction between EGFR and SOCS proteins(e.g., SOCS1 and SOCS3) (Xia et al., J. Biol. Chem., 277(34):30716-23,2002). The interaction between EGFR and SOCS proteins is furtherbelieved to stimulate the proteasomal degradation of the EGFR complexand/or induce degradation of EGFR-associated STAT proteins and/or blockEGFR from further recruitment and activation of STAT proteins (Xia etal., J. Biol. Chem., 277(34):30716-23, 2002). In each instance, the SOCSprotein (e.g., SOCS1 and/or SOCS3) interaction directly or indirectlyinhibits EGFR activity.

As demonstrated in this disclosure, epitopes present in the L2G Peptidesequence of EGFR are inaccessible to cognate RD-binding molecules (e.g.,antibodies) in some normal or neoplastic tissues. The accessibility ofsuch epitope is restored in tissues that lack proteins that normallybind the EGFR regulatory domain and the EGFR inhibitory subdomain.Hence, the disclosed L2G Peptide and other RDPs derived therefrom areuseful, at least, to make RD-binding molecules (such as antibodies,antibody fragments, scaffold polypeptides including antibody bindingdomains and aptamers) that expose the structural and correspondingfunctional states of EGFR.

In one embodiment, a disclosed RDP is the L2G Peptide, which has thesequence LDNPDYQQDFFPKEAKPNG (SEQ ID NO: 2). Also contemplated in someembodiments are immunogenic fragments of the L2G Peptide, whichfragments can be useful for producing a disclosed RD-binding molecule.For example, as demonstrated in Example 4, at least the subsequenceQQDFFPK (residues 7-13 of SEQ ID NO: 2) is sufficient to produce adisclosed RD-binding molecule (e.g., monoclonal antibody). Thus, in someembodiments, an immunogenic fragment of SEQ ID NO: 2 is at least 7contiguous residues of SEQ ID NO: 2 and includes the sequence QQDFFPK(residues 7-13 of SEQ ID NO: 2). In more specific embodiments animmunogenic fragment of SEQ ID NO: 2 is between 7 and 18 contiguousresidues of SEQ ID NO: 2 and includes the sequence QQDFFPK (residues7-13 of SEQ ID NO: 2). In other specific embodiments an immunogenicfragment of SEQ ID NO: 2 is between 10 and 18 contiguous residues of SEQID NO: 2 and includes the sequence QQDFFPK (residues 7-13 of SEQ ID NO:2). In each instance an immunogenic fragment of SEQ ID NO: 2 has afunction described herein (see, e.g., Abbreviations and Terms) orotherwise known in the art.

Further, at least because the subsequence QQDFFPK (residues 7-13 of SEQID NO: 2) is sufficient to produce a disclosed antigen-binding molecule,other RDP embodiments have the consensus sequence X1-6QQDFFPKX7-12 (SEQID NO: 6), where X1 through X12 are any amino acid. In more specificembodiments, an L2G peptide has the sequence X1-6QQDFFPKX7-12 (SEQ IDNO: 6), where X1 through X12 are any conservative substitution (e.g.,very highly conserved substitution, highly conserved substitution orconserved substitution) of the corresponding amino acid residue in SEQID NO: 2. Exemplary conservative amino acid substitutions are set forthin the following table:

Very Highly- Highly Conserved Original Conserved Substitutions (fromConserved Substitutions Residue Substitutions the Blosum90 Matrix) (fromthe Blosum65 Matrix) Ala Ser Gly, Ser, Thr Cys, Gly, Ser, Thr, Val ArgLys Gln, His, Lys Asn, Gln, Glu, His, Lys Asn Gln; His Asp, Gln, His,Lys, Arg, Asp, Gln, Glu, His, Ser, Thr Lys, Ser, Thr Asp Glu Asn, GluAsn, Gln, Glu, Ser Cys Ser None Ala Gln Asn Arg, Asn, Glu, His, Arg,Asn, Asp, Glu, His, Lys, Met Lys, Met, Ser Glu Asp Asp, Gln, Lys Arg,Asn, Asp, Gln, His, Lys, Ser Gly Pro Ala Ala, Ser His Asn; Gln Arg, Asn,Gln, Tyr Arg, Asn, Gln, Glu, Tyr Ile Leu; Val Leu, Met, Val Leu, Met,Phe, Val Leu Ile; Val Ile, Met, Phe, Val Ile, Met, Phe, Val Lys Arg;Gln; Glu Arg, Asn, Gln, Glu Arg, Asn, Gln, Glu, Ser, Met Leu; Ile Gln,Ile, Leu, Val Gln, Ile, Leu, Phe, Val Phe Met; Leu; Tyr Leu, Trp, TyrIle, Leu, Met, Trp, Tyr Ser Thr Ala, Asn, Thr Ala, Asn, Asp, Gln, Glu,Gly, Lys, Thr Thr Ser Ala, Asn, Ser Ala, Asn, Ser, Val Trp Tyr Phe, TyrPhe, Tyr Tyr Trp; Phe His, Phe, Trp His, Phe, Trp Val Ile; Leu Ile, Leu,Met Ala, Ile, Leu, Met, Thr

Some exemplary RDPs having the consensus sequence X1-6QQDFFPKX7-12, (SEQID NO: 6) wherein any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 or all 12 ofresidues X1 through X12 will have conservative amino acid changes (suchas, very highly conserved substitutions, highly conserved substitutionsor conserved substitutions) as compared to SEQ ID NO: 2 and, asapplicable, the remaining residues will have no change as compared toSEQ ID NO: 2.

In other embodiments, a RDP is a sequence variant of an L2G Peptide thathas at least 99%, at least 98%, at least 95%, at least 92%, at least90%, at least 85%, at least 80%, at least 75%, at least 70%, at least65%, or at least 60% amino acid sequence identity to the amino acidsequence set forth in SEQ ID NO: 2. “Sequence identity” is a phrasecommonly used to describe the similarity between two amino acidsequences (or between two nucleic acid sequences). Sequence identitytypically is expressed in terms of percentage identity; the higher thepercentage, the more similar the two sequences.

Methods for aligning sequences for comparison and determining sequenceidentity are well known in the art. Various programs and alignmentalgorithms are described in: Smith and Waterman, Adv. Appl. Math.,2:482, 1981; Needleman and Wunsch, J. Mol. Biol., 48:443, 1970; Pearsonand Lipman, Proc. Natl. Acad. Sci. USA, 85:2444, 1988; Higgins andSharp, Gene, 73:237-44, 1988; Higgins and Sharp, CABIOS, 5:151-3, 1989;Corpet et al., Nucleic Acids Research, 16:10881-90, 1988; Huang, et al.,Computer Applications in the Biosciences, 8:155-65, 1992; Pearson etal., Methods in Molecular Biology, 24:307-331, 1994; Tatiana et al.,FEMS Microbiol. Lett., 174:247-50, 1999. Altschul et al. present adetailed consideration of sequence-alignment methods and homologycalculations (J. Mol. Biol., 215:403-10, 1990).

The National Center for Biotechnology Information (NCBI) Basic LocalAlignment Search Tool (BLAST™, Altschul et al., J. Mol. Biol.,215:403-10, 1990) is publicly available from several sources, includingthe National Center for Biotechnology Information (NCBI, Bethesda, Md.)and on the Internet, for use in connection with the sequence-analysisprograms blastp, blastn, blastx, tblastn and tblastx. A description ofhow to determine sequence identity using this program is available onthe internet under the help section for BLAST™.

For comparisons of amino acid sequences of greater than about 15 aminoacids, the “Blast 2 sequences” function of the BLAST™ (Blastp) programis employed using the default BLOSUM62 matrix set to default parameters(cost to open a gap [default=5]; cost to extend a gap [default=2];penalty for a mismatch [default=3]; reward for a match [default=1];expectation value (E) [default=10.0]; word size [default=3]; and numberof one-line descriptions (V) [default=100]. When aligning short peptides(fewer than around 15 amino acids), the alignment should be performedusing the Blast 2 sequences function “Search for short nearly exactmatches” employing the PAM30 matrix set to default parameters (expectthreshold=20000, word size=2, gap costs: existence=9 and extension=1)using composition-based statistics.

Any disclosed sequence variant of a L2G Peptide (whether it is a varianthaving one or more conservative amino acid substitutions as compare toSEQ ID NO: 2 or a variant having a disclosed percentage sequenceidentity to SEQ ID NO: 2), at least, is immunogenic (alone or whencouple to a carrier molecule) and, e.g., capable of eliciting productionof a RD-binding molecule (such as a monoclonal antibody).

VI. EGFR Regulatory Domain-Binding Molecules

This disclosure also concerns EGFR-specific RD-binding molecules.RD-binding molecules are a species of antigen-binding molecules thatspecifically bind epitopes in the EGFR regulatory domain (whichcorresponds to residues 980-1210 of SEQ ID NO: 1), such as epitopes inthe EGFR inhibitory subdomain (which corresponds to residues 1138-1196of SEQ ID NO: 1).

Exemplary RD-binding molecules include RD-binding molecules specific fora L2G Peptide, or any epitope contained therein, or RD-binding moleculesspecific for a L2G Peptide sequence in EGFR (including RD-bindingmolecules that are competitively inhibited from binding EGFR by a L2GPeptide or fragment thereof), RD-binding molecules that recognizeparticular structural states of EGFR (for instance, RD-binding moleculesspecific for epitopes masked by EGFR protein-protein interactions),and/or RD binding molecules that recognize particular regulated statesof EGFR (for instance, RD-binding molecules specific for epitopescontained with an EGFR inhibitory subdomain and which recognize thebinding of a negative regulatory molecule (e.g., SOCS1 or SOCS2) toEGFR.

RD-binding molecules include, for example, antibodies or functionalfragments or recombinant derivatives thereof, aptamers, mirror-imageaptamers, or engineered nonimmunoglobulin binding proteins based on anyone or more of the following scaffolds: fibronectin (e.g., ADNECTINS™ ormonobodies), CTLA-4 (e.g., EVIBODIES™), tendamistat (e.g., McConnell andHoess, J. Mol. Biol., 250:460-470, 1995), neocarzinostatin (e.g., Heydet al., Biochem., 42:5674-5683, 2003), CBM4-2 (e.g., Cicortas-Gunnarssonet al., Protein Eng. Des. Sel., 17:213-221, 2004), lipocalins (e.g.,ANTICALINS™; Schlehuber and Skerra, Drug Discov. Today, 10:23-33, 2005),T-cell receptors (e.g., Chlewicki et al., J. Mol. Biol., 346:223-239,2005), protein A domain (e.g., AFFIBODIES™; Engfeldt et al., Chem BioChem, 6:1043-1050, 2005), Im9 (e.g., Bernath et al., J. Mol. Biol.,345:1015-1026, 2005), ankyrin repeat proteins (e.g., DARPins; Amstutz etal., J. Biol. Chem., 280:24715-24722, 2005), tetratricopeptide repeatproteins (e.g., Cortajarena et al., Protein Eng. Des. Sel., 17:399-409,2004), zinc finger domains (e.g., Bianchi et al., J. Mol. Biol.,247:154-160, 1995), pVIII (e.g., Petrenko et al., Protein Eng.,15:943-950, 2002), GCN4 (Sia and Kim, Proc. Natl. Acad. Sci. USA,100:9756-9761, 2003), avian pancreatic polypeptide (APP) (e.g., Chin etal., Bioorg. Med. Chem. Lett., 11:1501-1505, 2001), WW domains, (e.g.,Dalby et al., Protein Sci., 9:2366-2376, 2000), SH3 domains (e.g.,Hiipakka et al., J. Mol. Biol., 293:1097-1106, 1999), SH2 domains(Malabarba et al., Oncogene, 20:5186-5194, 2001), PDZ domains (e.g.,TELOBODIES™; Schneider et al., Nat. Biotechnol., 17: 170-175, 1999),TEM-1 β-lactamase (e.g., Legendre et al., Protein Sci., 11: 1506-1518,2002), green fluorescent protein (GFP) (e.g., Zeytun et al., Nat.Biotechnol., 22:601, 2004), thioredoxin (e.g., peptide aptamers; Lu etal., Biotechnol., 13:366-372, 1995), Staphylococcal nuclease (e.g.,Norman, et al., Science, 285:591-595, 1999), PHD fingers (e.g., Kwan etal., Structure, 11:803-813, 2003), chymotrypsin inhibitor 2 (CI2) (e.g.,Karlsson et al., Br. J. Cancer, 91:1488-1494, 2004), bovine pancreatictrypsin inhibitor (BPTI) (e.g., Roberts, Proc. Natl. Acad. Sci. USA,89:2429-2433, 1992) and many others (see review by Binz et al., Nat.Biotechnol., 23(10): 1257-1268, 2005 and supplemental materials).

In one embodiment, a disclosed RD-binding molecule is an aptamer.Aptamers include single-stranded nucleic acid molecules (such as, DNA orRNA) that assume a specific, sequence-dependent shape and binds to atarget protein with high affinity and specificity. Aptamers generallycomprise fewer than 100 nucleotides, fewer than 75 nucleotides, or fewerthan 50 nucleotides. In another embodiment, a disclosed RD-bindingmolecule is a mirror-image aptamer (also called a SPIEGELMER™).Mirror-image aptamers are high-affinity L-enantiomeric nucleic acids(for example, L-ribose or L-2′-deoxyribose units) that display highresistance to enzymatic degradation compared with D-oligonucleotides(such as, aptamers). The target binding properties of aptamers andmirror-image aptamers are designed by an in vitro-selection processstarting from a random pool of oligonucleotides, as described forexample, in Wlotzka et al., Proc. Natl. Acad. Sci. 99(13):8898-902,2002.

In another example, an aptamer is a peptide aptamer that binds to atarget protein (e.g., an EGFR RD) with high affinity and specificity.Peptide aptamers include a peptide loop (e.g., which is specific for thetarget) attached at both ends to a protein scaffold. This doublestructural constraint greatly increases the binding affinity of thepeptide aptamer to levels comparable to an antibody's (nanomolar range).The variable loop length is typically 8 to 20 amino acids (e.g., 8 to 12amino acids), and the scaffold may be any protein which is stable,soluble, small, and non-toxic (e.g., thioredoxin-A, stefin A triplemutant, green fluorescent protein, eglin C, and cellular transcriptionfactor Sp1). Peptide aptamer selection can be made using differentsystems, such as the yeast two-hybrid system (e.g., Gal4yeast-two-hybrid system) or the LexA interaction trap system.

Disclosed RD-binding molecules also include antibodies. The term“antibody” refers to an immunoglobulin molecule (or combinationsthereof) that specifically binds to, or is immunologically reactivewith, a particular antigen, and includes polyclonal, monoclonal,genetically engineered and otherwise modified forms of antibodies,including but not limited to chimeric antibodies, humanized antibodies,heteroconjugate antibodies (e.g., bispecific antibodies, diabodies,triabodies, and tetrabodies), single chain Fv antibodies (scFv),polypeptides that contain at least a portion of an immunoglobulin thatis sufficient to confer specific antigen binding to the polypeptide, andantigen binding fragments of antibodies, including, e.g., Fab′, F(ab′)2,Fab, Fv, rIgG, or complementarity determining region (CDR) fragments.

A Fab fragment is a monovalent fragment consisting of the VL, VH, CL andCH1 domains; a F(ab′)₂ fragment is a bivalent fragment comprising twoFab fragments linked by a disulfide bridge at the hinge region; an Fdfragment consists of the VH and CHI domains; an Fv fragment consists ofthe VL and VH domains of a single arm of an antibody; and a dAb fragmentconsists of a VH domain (see, e.g., Ward et al., Nature 341:544-546,1989). A single-chain antibody (scFv) is an antibody in which a VL andVH region are paired to form a monovalent molecule via a syntheticlinker that enables them to be made as a single protein chain (see,e.g., Bird et al., Science, 242: 423-426, 1988; Huston et al., Proc.Natl. Acad. Sci. USA, 85:5879-5883, 1988). Diabodies are bivalent,bispecific antibodies in which VH and VL domains are expressed on asingle polypeptide chain, but using a linker that is too short to allowfor pairing between the two domains on the same chain, thereby forcingthe domains to pair with complementary domains of another chain andcreating two antigen binding sites (see, e.g., Holliger et al., Proc.Natl. Acad. Sci. USA, 90:6444-8, 1993; Poljak et al., Structure,2:1121-3, 1994). A chimeric antibody is an antibody that contains one ormore regions from one antibody and one or more regions from one or moreother antibodies. An antibody may have one or more binding sites. Ifthere is more than one binding site, the binding sites may be identicalto one another or may be different. For instance, a naturally occurringimmunoglobulin has two identical binding sites, a single-chain antibodyor Fab fragment has one binding site, while a “bispecific” or“bifunctional” antibody has two different binding sites.

As discussed above, exemplary RD-binding molecules recognize particularregulated or structural states of EGFR. For example, a disclosedRD-binding molecule can detect the masking (or unmasking) of an epitopein the EGFR regulatory domain (residues 980-1210 of SEQ ID NO: 1). Suchepitope masking (or unmasking) can result, for instance, from aprotein-protein interaction between EGFR and another cellular protein,such as a SOCS protein (e.g., SOCS1 or SOCS3); wherein the binding ofthe cellular protein to EGFR masks the epitope and the disassociation(or lack of association) of the two proteins unmasks the epitope.

In some examples, RD-binding molecules, such as antibodies (e.g.,monoclonal antibody) or fragments thereof, are characterized by specificbinding to any one or more EGFR RDPs disclosed herein (see, e.g.,Section V). In other examples, RD-binding molecules, such as antibodies(e.g., monoclonal antibody) or fragments thereof, specifically bind toamino acid residues of EGFR that correspond to the sequence(s) ofdisclosed RDPs (see, e.g., Section V). In still other examples,RD-binding molecules, such as antibodies (e.g., monoclonal antibody) orfragments thereof, specifically bind to the EGFR regulatory domain or tothe EGFR inhibitory subdomain and such specific binding is competitivelyinhibited by any one or more EGFR RDPs disclosed herein (see, e.g.,Section V). Other examples involve RD-binding molecules (such asantibodies (e.g., monoclonal antibody) or fragments thereof) thatspecifically bind to the EGFR regulatory domain or to the EGFRinhibitory subdomain; wherein such specific binding is competitivelyinhibited by a SOCS protein, such as SOCS1 or SOCS3 (or a fragment of aSOCS protein that binds to the regulatory domain of EGFR (for example, aregion of the regulatory domain including a phosphorylated Tyrresidue)).

In one embodiment, a RD-binding molecule is a rabbit monoclonalantibody. In one particular embodiment, a RD-binding molecule is arabbit monoclonal antibody deposited at ATCC Accession No. ______. Inanother particular embodiment, a RD-binding molecule is rabbitmonoclonal antibody clone 5B7, which is commercially available fromVentana Medical Systems (Tucson, Ariz.; product number 790 4347).

In some examples, a RD-binding molecule (such as an antibody (e.g.,monoclonal antibody) or fragments thereof) has an equilibrium constant(K_(d)) of 1 nM or less. For example, RD-binding molecules are providedthat bind the regulatory domain (or inhibitory subdomain) of EGFR with abinding affinity of at least about 0.1×10⁻⁸ M, at least about 0.3×10⁻⁸M,at least about 0.5×10⁻⁸M, at least about 0.75×10⁻⁸ M, at least about1.0×10⁻⁸ M, at least about 1.3×10⁻⁸ M at least about 1.5×10⁻⁸M, or atleast about 2.0×10⁻⁸ M.

A disclosed RD-binding molecule, such as an antibody (e.g., monoclonalantibody) or fragments thereof, optionally can be directly labeled witha detectable moiety. Useful detection agents include fluorescentcompounds (including fluorescein, fluorescein isothiocyanate, rhodamine,5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin, lanthanidephosphors, or the cyanine family of dyes (such as Cy-3 or Cy-5) and thelike); bioluminescent compounds (such as luciferase, green fluorescentprotein (GFP), or yellow fluorescent protein); enzymes that can producea detectable reaction product (such as horseradish peroxidase,β-galactosidase, luciferase, alkaline phosphatase, or glucose oxidaseand the like), or radiolabels (such as ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc,¹¹¹In, ¹²⁵I, or ¹³¹I).

A. Making of Exemplary RD-Binding Antibodies

Methods of making EGFR RD-binding molecules are well known in the art.The method used will depend upon the nature of the desired RD-bindingmolecules; for instance peptide-based RD-binding molecules that are notnecessarily immunoglobulin in origin can be made using methods that aresimilar to phage display methods. One such method is described inSzardenings, J. Recept. Signal Transduct. Res., 23:307-309, 2003.

Methods of generating antibodies (such as monoclonal or polyclonalantibodies) are well established in the art (for example see Harlow andLane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,New York, 1988). For example RDPs or RDPs conjugated to carriermolecules (or nucleic acids encoding such RDPs or conjugated RDPs) canbe injected into non-human mammals (such as mice or rabbits), followedby boost injections, to produce an antibody response. Serum isolatedfrom immunized animals may be isolated for the polyclonal antibodiescontained therein, or spleens from immunized animals may be used for theproduction of hybridomas and monoclonal antibodies.

In one example, monoclonal antibody to epitopes in RDPs can be preparedfrom murine hybridomas according to the classical method of Kohler andMilstein (Nature, 256:495, 1975) or derivative methods thereof. Briefly,a mouse (such as Balb/c) is repetitively inoculated with a fewmicrograms of the selected RDP (such as SEQ ID NO: 2) or carrierconjugate thereof over a period of a few weeks. The mouse is thensacrificed, and the antibody-producing cells of the spleen isolated. Thespleen cells are fused by means of polyethylene glycol with mousemyeloma cells, and the excess unfused cells destroyed by growth of thesystem on selective media comprising aminopterin (HAT media). Thesuccessfully fused cells are diluted and aliquots of the dilution placedin wells of a microtiter plate where growth of the culture is continued.Antibody-producing clones are identified by detection of antibody in thesupernatant fluid of the wells by immunoassay procedures, such as ELISA,as originally described by Engvall (Enzymol., 70:419, 1980), andderivative methods thereof. Selected positive clones can be expanded andtheir monoclonal antibody product harvested for use.

In another example, monoclonal antibody to epitopes in RDPs can beprepared from rabbit hybridomas as described in U.S. Pat. No. 7,148,332,5,675,063, or 4,859,595.

In yet another example, monoclonal antibodies to epitopes of the EGFRinhibitory domain can be prepared by repetitively inoculating anon-human mammal (such as a mouse or rabbit) with one or more plasmidsencoding a disclosed RDP (such as a plasmid encoding SEQ ID NO: 2). Forexample, pcDNA3 (Invitrogen, Carlsbad, Calif.) or a vector derived therefrom, can be manipulated using standard molecular biology methods toinclude a coding sequence for a disclosed RDP (e.g., SEQ ID NO: 2). Inone exemplary method, Balb/c mice (6-8 weeks old) are immunized threetimes with the appropriate plasmid (20 μg in phosphate-buffered saline),and one boost can be given with cells before fusion. Mice can beinjected three times intradermally into the base of the tail on days 0,10, and 20 using an insulin syringe with a 28-gauge needle attached.Serum can be drawn on days 30 and 45 for evaluation of the anti-serumtiter. To boost the immunized mice, cells expressing the desired plasmidare injected (for example on day at least 50). These injections can beintravenous and intraperitoneal. Spleens are harvested about 80-90 hoursafter the last cell boost for cell fusion.

Cell fusions of the splenocytes can be performed according to theprotocol of Oi and Herzenberg (Selected Methods in Cellular Immunology,Freeman Press, San Francisco, 1980). Splenocytes and SP2/0 cells aremixed, for example at a 4:1 ratio. The mixed cells are centrifuged andthe cell pellet resuspended in polyethylene glycol (such as 40%-50%(w/v) polyethylene glycol) and appropriate medium. The resultingsuspension is centrifuged and the cell pellet resuspended in HAT medium,and seeded in 96-well plates at 100 μl/well (2.5×10⁵ cells/well) andcultured in a CO₂ incubator. On the day after fusion, 100 μl of freshHAT medium containing 500 μg/ml geneticin (Invitrogen) is added. On days4 and 7, half of the spent medium is replaced by fresh HAT mediumcontaining 250 μg/ml geneticin. On day 8, the growth of the hybridoma ineach well is checked under a microscope. mAb production in culturesupernatants can be assayed on day 10 by ELISA assay or days 9 and 10 byFACS sorter. Positive clones can be expanded and the specific hybridomascloned by a limiting dilution method.

In addition, protocols for producing humanized forms of monoclonalantibodies and fragments of monoclonal antibodies are known in the art(see, e.g., U.S. Pat. Nos. 6,054,297, 6,407,213, 6,639,055, 6,800,738,and 6,719,971 and U.S. Pat. Appl. Pub. Nos. 2005/0033031, and2004/0236078). Similarly, methods for producing single chain antibodieshave been described and can be useful for the making of RD-bindingmolecules disclosed herein (see, Buchner et al., Anal. Biochem.205:263-270, 1992; Pluckthun, Biotechnology 9:545, 1991; Huse et al.,Science 246:1275, 1989 and Ward et al., Nature 341:544, 1989).

B. Making of Exemplary RD-Binding Aptamers

Methods of generating aptamers (e.g., DNA or RNA aptamers) are wellestablished in the art. For example, with knowledge of an RD sequence(see Section V) aptamers can be selected that bind to an RD amino acidsequence.

In one example, DNA or RNA aptamers are selected using the in vitromethod SELEX (systematic evolution of ligands by exponentialenrichment), for example using the method of Fitzwater and Polisky(Methods Enzymol., 267:275-301, 1996). Such a method can be used toidentify aptamers that bind with high specificity to a RD. The SELEXprocedure is usually initiated with an RNA or DNA library containingabout 10¹⁴-10⁵ random oligonucleotide sequences. In a fully randomizedoligonucleotide library, each molecule will exhibit a unique tertiarystructure that will be dependent on the nucleotide sequence of thatmolecule. The binding affinity of the oligonucleotide for a particularRD will be determined by the fit between moieties on the surface of theoligonucleotide and epitopes on the target RD. By starting from alibrary of vast diversity, aptamers of nanomolar or subnanomolaraffinity for the target RD with selectivity for that RD over other RDswith a high degree of structural homology can be identified. Forexample, RD peptides (or portion thereof, such as 3 to 20 amino acids ofa target RD, for example an epitope) can be attached to a surface (suchas a 96-well or other multi-well microtiter plate). The library ofnucleic acid molecules can be added to the bound peptide underconditions that permit members of the library to bind to the peptide(e.g., by incubating at 37° C. for 30 minutes). Unbound members of thelibrary are washed away, and then bound members of the library areeluted (e.g., by incubating at 95° C. for 10 minutes). Reversetranscription is performed (if the aptamers are RNA), followed bypolymerase chain reaction and transcription to generate nucleic acidsfor the next round of SELEX. The dissociation constant (Kd) forresulting selected aptamer can be determined using routine methods.Aptamers with high affinity for the desired RD can be selected, such asa Kd of less than 100 nM, such as less than 50 nM, less than 10 nM, orless than 1 nM (for example 0.1 to 50 nM). Aptamers can be modified toincrease their half-life, for example modified with2′-fluorine-substituted pyrimidines, 2′-ribo purines, polyethyleneglycol (PEG) linkage, and the like.

In one example, peptide aptamers are selected using a yeast two hybridsystem, for example using the method of de Chassey et al. (Molecular &Cellular Proteomics 6:451-9, 2007). Reviews are provided, for example,in Borghouts et al. (Comb. Chem. High Throughput Screen. 11: 135-45,2008) and Buerger et al. (J. Cancer Res. Clin. Oncol. 129:669-75, 2003).Such a method can be used to identify peptide aptamers that bind withhigh specificity to a RD. A peptide aptamer library of high complexityis screened, such as 20mer or 8-12mer libraries. The library may or maynot be based on information known about the sequence of the RD. In oneexample, the library includes oligonucleotides encoding variant peptidesbased on the amino acid sequence of the target RD. The library includesoligonucleotides encoding the variable peptides inserted into a vectorencoding the scaffold protein (e.g. thioredoxin). When expressed, “prey”peptide sequences are embedded in the scaffold protein. A nucleic acidsequence or vector encoding the “bait” target RD fused to atranscription module (e.g., Gal4 or LexA) is expressed in the cells(e.g., yeast) along with the “prey” coding sequences.

If the yeast-two-hybrid system is used, the “prey” peptide aptamer canbe fused to Gal4-transactivation domain (Gal4-AD) and can also include anuclear localization signal and an HA tag for detection. Exemplaryvectors that can be used to express the peptide aptamer include pRS424,pAD-Trx, pGAD424; pGAD-T7, pACT2, and pAD-Gal4-2.1. A vector encodingthe “bait” target protein fused to the Gal4 DNA binding domain isexpressed in yeast along with the “prey” coding sequences. Exemplaryvectors that can be used to express the “bait” peptide include pPC97,pLex9, pGBK-T7, and pDB-Gal4Cam. In some examples, the reporter yeaststrain into which prey and bait vectors are introduced include His3,Ade2, Ura3 and LacZ genes under the control of a Gal upstream activatingsequences to permit selection of clones where the bait and preyspecifically bind. To select for desired peptide aptamers, transformedyeast cells are placed on media lacking histidine, adenine, or uracil.β-gal assays can be performed to quantify binding between identifiedaptamers and the target. To increase selection stringency, the amount of3-AT inhibitor (e.g., 10-100 mM) can be increased. Cells that growindicate the presence of peptide aptamer binding to the target RD.

If the LexA interaction trap system is used, a vector encoding the“prey” peptide aptamer fused to B42 or B112 acid transactivation domaincan be used. Exemplary vectors that can be used to express the peptideaptamer include pWP1, pWP2, pJG4-5, pJM1, pHA3). A vector encoding the“bait” target protein fused to the DNA binding domain of the LexArepressor is expressed in yeast along with the “prey” coding sequences.An exemplary vector that can be used to express the “bait” peptideincludes pEG202. Expression of the prey vector is induced if galactoseis present in the growth medium. To select for desired peptide aptamers,transformed yeast cells are placed on media with galactose. Interactionsbetween bait protein and peptide aptamer are detected on galactoseplates that lack leucine. Cells that grow indicate the presence ofpeptide aptamer binding to the target RD.

Clones indicated to carry the desired protein aptamer that binds to theRD can be selected, and the vector encoding the aptamer isolated andcloned using standard recombinant technology.

VII. Kits

Any of the RD-binding molecules described in this disclosure can besupplied in the form of a kit useful, at least, for performing themethods described herein. In one embodiment of such a kit, anappropriate amount of at least one RD-binding molecule (e.g., monoclonalantibody (such as clone 5B7) or fragment thereof) is provided in one ormore containers. In other embodiments, at least one RD-binding molecule(e.g., monoclonal antibody (such as clone 5B7) or fragment thereof) maybe provided suspended in an aqueous solution or as a freeze-dried orlyophilized powder, for instance. The container(s) in which the at leastone RD-binding molecule (e.g., monoclonal antibody (such as clone 5B7)or fragment thereof) is supplied can be any conventional container thatis capable of holding the supplied form, for instance, microfuge tubes,ampoules, or bottles. The amount of RD-binding molecule (e.g.,monoclonal antibody (such as clone 5B7) or fragment thereof) suppliedcan be any appropriate amount, such as from about 1 to about 5 μg/ml.

In other embodiments, control slides upon which are mounted one or moretissue or cell preparations (e.g., xenografts, cell pellets, or clottedcells) that may serve as positive and/or negative controls for aRD-binding molecule (e.g., monoclonal antibody (such as clone 5B7) orfragment thereof) may be provided in an appropriate and separatecontainer. In some instances, A431, DU145, and/or Caski cells (orxenografts prepared therewith) may serve as a positive control. In otherinstances, MCF-7 cells (or xenografts prepared therewith) may serve as anegative control.

Other kit embodiments will include means for detection of the RD-bindingmolecule, such as secondary antibodies (e.g., goat anti-rabbitantibodies or rabbit anti-mouse antibodies). In some such instances, thesecondary antibody will be directly labeled with a detectable moiety (asdescribed elsewhere in this disclosure). In other instances, the primaryor secondary (or higher-order) antibody will be conjugated to a hapten(such as biotin, DNP, and/or FITC), which is detectable by a detectablylabeled cognate hapten-binding molecule (e.g. streptavidin (SA)-horseradish peroxidase, SA-alkaline phosphatase, and/or SA-QDot™). Some kitembodiments may include colorimetric reagents (e.g., DAB, and/or AEC) insuitable containers to be used in concert with primary or secondary (orhigher-order) antibodies that are labeled with enzymes for thedevelopment of such calorimetric reagents.

In one embodiment, a kit includes instructional materials disclosingmethods of use of the kit contents (e.g., RD-binding molecule) in adisclosed method. The instructional materials may be written, in anelectronic form (e.g. computer diskette or compact disk) or may bevisual (e.g. video files). The kits may also include additionalcomponents to facilitate the particular application for which the kit isdesigned. Thus, for example, the kits may additionally include buffersand other reagents routinely used for the practice of a particularmethod. Such kits and appropriate contents are well known to those ofskill in the art.

The following examples are provided to illustrate certain particularfeatures and/or embodiments. These examples should not be construed tolimit the invention to the particular features or embodiments described.

EXAMPLES Example 1 An Exemplary Monoclonal Antibody Specific for EGFRRegulatory Domain

This Example describes an exemplary RD-binding molecule; moreparticularly a monoclonal antibody that binds an epitope in the EGFRinhibitory subdomain. This antibody has the added advantage that it willidentify not only full-length EGFR, but also truncated mutant forms ofEGFR, which have been shown to be constitutively activated (Pedersen etal., Ann. Oncol., 12(6):745-60, 2001).

A computer program (DNASTAR™, Madison, Wis.) was used for the selectionof immunogenic peptide sequences within the EGFR intracellular domain.The program examined the input protein sequence for short (e.g., lessthan 20 contiguous amino acids) sequences that likely had a highprobability for producing an antibody response in animals immunized withimmunogens including such short sequences.

One identified short sequence was LDNPDYQQDFFPKEAKPNG (L2G Peptide; SEQID NO: 2), which, by computer analysis, had high antigenicity, highhydrophilic regions, and high surface probability regions. This aminoacid sequence was selected and a corresponding peptide was synthesizedusing a commercially available service (Anaspec, San Jose, Calif.).

The synthesized peptide was conjugated to Keyhole Limpet Hemocyanin(KLH) using standard methods. Rabbits were immunized with theKLH-peptide conjugate by a commercially available service (StrategicDiagnostics, Inc. Newark, Del.).

Rabbit sera containing antibodies specific for the L2G Peptide wereidentified by ELISA assay. The animal with the strongest serum titer wasselected for a splenectomy. The viable spleen was shipped to Epitomics,Inc (Burlingame, Calif.) overnight where the immunized spleen cells wereprepared for fusion with an immortalized cell line (240E-w) asdescribed, e.g., in U.S. Pat. No. 5,675,063 or European Pat. No.EP0815213B1.

Hybridoma supernatants were tested by ELISA assay for the presence ofantibodies specific for the L2G Peptide. One hybridoma was selectedbased on a relatively high antibody titer in the correspondingsupernatant. The specificity of antibodies produced by the selectedhybridoma cell line was confirmed by immunohistochemistry (IHC) testingon known EGFR-positive tissues (including squamous cell carcinoma of thelung, colon adenocarcinomas, and normal skin). The hybridoma cell linedelivered by the manufacturer was subcloned to homogeneity to isolate ahigh-producing hybridoma clone designated 5B7.

A Western blot analysis was performed to ensure the specificity of clone5B7. Total protein lysates were prepared from A431 cells, which areknown to express high levels of EGFR on their cell surface, and fromBT474 cells, which are negative for EGFR, but which express related EGFRfamily members, EGFR2 and EGFR3. As shown in FIG. 4, clone 5B7 and amouse monoclonal antibody specific for the EGFR external domain (clone3C6) recognized the same 170 kDa band, which is consistent with the sizeof the EGFR protein. The lack of staining of the BT474 cell lysates forboth antibodies indicated that neither antibody cross reacted with EGFRfamily members that have conserved homology. Thus, the 3C6 and 5B7antibodies were specific for EGFR.

Example 2 Exemplary Methods for Immunohistochemical Staining of Tissuewith EGFR-Specific Antibodies

Immunohistochemistry is the well-known method and variations on suchmethods are readily determined with routine experimentation by those ofordinary skill in the art (see, e.g., Dabbs, DiagnosticImmunohistochemistry, Churchill Livingstone, 2002). Exemplary methodsfor detecting in FFPE tissue by manual IHC an EGFR RD-binding molecule(e.g., monoclonal antibody clone 5B7) or an antigen-binding moleculespecific for the EGFR extracellular domain (e.g., monoclonal antibodyclone 3C6) are provided in the following table:

Step # Manual IHC Assay 0 Fresh tissue is placed in a fixative (such as,10% neutral buffered formalin) for approximately 12-48 hours at roomtemperature. Then, the tissue is dehydrated through graded alcohols(e.g., 50% to 70% to 90% to 95% to 100% EtOH) for 1-2 hours at eachgrade, and infiltrated with a clearing reagent (such as, xylene) for 3-5hours at room temperature. The cleared tissue is placed in melted(approximately 63 degrees C) paraffin for 3-6 hours. Samples are removedand embedded in paraffin blocks for subsequent microtome sectioning.3-10 μm sections are cut and placed on glass slides. 1 Deparaffinizetissue sections in xylene; then, rehydrate through graded alcohols todistilled water. 2 Place tissue sections in 0.5% v/v hydrogenperoxide/methanol for approximately 10 minutes. 3 Pretreat slides forantigen retrieval using an appropriate method (e.g., high-temperatureantigen unmasking, trypsin, etc.) if required. 4 Wash slides withdistilled water for approximately 5 minutes. 5 Wash slides in salinebuffer (e.g., PBS, TBS) for 5 minutes. 6 Cover tissue sections withblocking reagent (e.g., 10% v/v normal rabbit serum in buffer) forapproximately 10 minutes. 7 Remove excess blocking reagent and replacewith primary antibody (e.g., rabbit monoclonal antibody or mousemonoclonal antibody) diluted in blocking reagent as required forapproximately 60 minutes at 25° C. or overnight at 4° C. 8 Wash twice inbuffer for approximately 5 minutes per wash. 9 Remove excess buffer andincubate tissue sections with biotinylated secondary antibody (e.g.,biotinylated rabbit anti-mouse antibody or biotinylated goat anti-rabbitantibody as appropriate for the subject primary antibody) diluted inblocking reagent for 30 minutes at 25° C. 10 Wash twice in buffer forapproximately 5 minutes per wash. 11 Remove excess buffer and incubatetissue sections with streptavidin-horse radish peroxidase (HRP)conjugate for 30 minutes at 25° C. 12 Wash twice in buffer forapproximately 5 minutes per wash. 13 Develop detectable color with3,3′-diaminobenzidine tetrahydrochloride (DAB) at room temperature forapproximately 5-10 minutes. 14 Rinse slides in water. 15 If desiredcounterstain with hematoxylin (e.g., Carson, Histotechnology: ASelf-Instructional Text, Chicago: ASCP Press, 1997). 16 Dehydrate, clearand mount coverslip on slides.

IHC for the detection antibodies specific for the EGFR regulatory domainalso can be performed on automated staining platforms, such as theBenchMark™ series instruments manufactured by Ventana Medical Systems(Tucson, Ariz.). An exemplary assay for the detection of a monoclonalantibody specific for the EGFR regulatory domain (e.g., clone 5B7) on aBenchMark™ series automated tissue stainer is described in the followingtable:

Step # Anti-EGFR Regulatory Domain Antibody Staining (Automated Assay) 1*** Select EZ Prep *** 2 *** Start Timed Steps *** 3 *** Mixers Off ***4 Warmup Slide to 75° C., and Incubate for 4 Minutes 5 Apply EZPrepVolume Adjust 6 Rinse Slide 7 Apply EZPrep Volume Adjust 8 Rinse Slide 9Apply EZPrep Volume Adjust 10 Apply Coverslip 11 Warmup Slide to 76° C.,and Incubate for 4 Minutes 12 Rinse Slide 13 Apply Depar Volume Adjust14 Apply Coverslip 15 Disable Slide Heater 16 *** Mixers On *** 17[Short - 8 Minute Conditioning] 18 Rinse Slide 19 Apply Long CellConditioner #1 20 Apply CC Coverslip Long 21 *** Select SSC Wash *** 22Warmup Slide to 95° C., and Incubate for 8 Minutes 23 [Mild - 30 MinuteConditioning] 24 Apply Cell Conditioner #1 25 Apply CC Medium CoverslipNo BB 26 Warmup Slide to 100° C., and Incubate for 4 Minutes 27 Apply CCMedium Coverslip No BB 28 Apply Cell Conditioner #1 29 Apply CC MediumCoverslip No BB 30 Apply Cell Conditioner #1 31 Apply CC MediumCoverslip No BB 32 Apply Cell Conditioner #1 33 Apply CC MediumCoverslip No BB 34 Apply Cell Conditioner #1 35 Apply CC MediumCoverslip No BB 36 Apply Cell Conditioner #1 37 Apply CC MediumCoverslip No BB 38 [Standard - 60 Minute Conditioning] 39 Apply CellConditioner #1 40 Apply CC Medium Coverslip No BB 41 Apply CellConditioner #1 42 Apply CC Medium Coverslip No BB 43 Apply CellConditioner #1 44 Apply CC Medium Coverslip No BB 45 Apply CellConditioner #1 46 Apply CC Medium Coverslip No BB 47 Apply CellConditioner #1 48 Apply CC Medium Coverslip No BB 49 Apply Short CellConditioner #1 50 Apply CC Medium Coverslip No BB 51 Apply CellConditioner #1 52 Apply CC Medium Coverslip No BB 53 Disable SlideHeater 54 Incubate for 8 Minutes 55 Rinse Slide With Reaction Buffer 56Adjust Slide Volume With Reaction Buffer 57 Apply Coverslip 58 RinseSlide With Reaction Buffer 59 Adjust Slide Volume With Reaction Buffer60 Apply Coverslip 61 *** Procedure Synchronization *** 62 Warmup Slideto 37° C., and Incubate for 4 Minutes 63 Rinse Slide With ReactionBuffer 64 Adjust Slide Volume With Reaction Buffer 65 Apply One Drop ofI-VIEW INHIBITOR, Apply Coverslip, and Incubate for 4 Minutes 66 RinseSlide With Reaction Buffer 67 Adjust Slide Volume With Reaction Buffer68 Apply Coverslip 69 Warmup Slide to 37° C., and Incubate for 4 Minutes70 Rinse Slide With Reaction Buffer 71 Adjust Slide Volume With ReactionBuffer 72 Apply Coverslip 73 Apply One Drop of Antibody (e.g., clone5B7), and Incubate for [0 Hr 16 Min] 74 Rinse Slide With Reaction Buffer75 Adjust Slide Volume With Reaction Buffer 76 Apply Coverslip 77 WarmupSlide to 37° C., and Incubate for 4 Minutes 78 Rinse Slide With ReactionBuffer 79 Adjust Slide Volume With Reaction Buffer 80 Apply One Drop ofI-VIEW BIOTIN Ig, Apply Coverslip, and Incubate for 8 Minutes 81 RinseSlide With Reaction Buffer 82 Adjust Slide Volume With Reaction Buffer83 Apply One Drop of I-VIEW SA-HRP, Apply Coverslip, and Incubate for 8Minutes 84 Rinse Slide With Reaction Buffer 85 Adjust Slide Volume WithReaction Buffer 86 Apply Coverslip 87 Rinse Slide With Reaction Buffer88 Adjust Slide Volume With Reaction Buffer 89 Apply One Drop of I-VIEWDAB and One Drop of I-VIEW H₂O₂, Apply Coverslip, Incubate for 8 Minutes90 Rinse Slide With Reaction Buffer 91 Adjust Slide Volume With ReactionBuffer 92 Apply One Drop of I-VIEW COPPER, Apply Coverslip, and Incubatefor 4 Minutes 93 Rinse Slide With Reaction Buffer 94 Adjust Slide VolumeWith Reaction Buffer 95 Apply One Drop of [HEMATOXYLIN II](Counterstain), Apply Coverslip, and Incubate for [4 Minutes] 96 RinseSlide With Reaction Buffer 97 Adjust Slide Volume With Reaction Buffer98 Apply Coverslip 99 Rinse Slide With Reaction Buffer 100 Adjust SlideVolume With Reaction Buffer 101 Apply One Drop of [BLUING REAGENT] (PostCounterstain), Apply Coverslip, and Incubate for [4 Minutes] 102 RinseSlide With Reaction Buffer 103 Apply Coverslip 104 Disable Slide Heater105 *** Select Optional Wash *** 106 *** Select SSC Wash *** 107 ***Start Timed Steps *** 108 Rinse Slide With Reaction Buffer

An exemplary assay for the detection of a monoclonal antibody specificfor the EGFR external domain (e.g., clone 3C6) on a BenchMark™ seriesautomated tissue stainer is described in the following table:

Step # Anti-EGFR External Domain Antibody Staining (Automated Assay) 1***** Select EZ Prep ***** 2 ***** Start Timed Steps ***** 3 *****Mixers Off ***** 4 Warm Slide to 75° C., and Incubate for 4 Minutes 5Apply EZPrep Volume Adjust 6 Rinse Slide 7 Apply EZPrep Volume Adjust 8Rinse Slide 9 Apply EZPrep Volume Adjust 10 Apply Coverslip 11 WarmSlide to 76° C., and Incubate for 4 Minutes 12 Rinse Slide 13 ApplyDepar Volume Adjust 14 Apply Coverslip 15 Disable Slide Heater 16 *****Mixers On ***** 17 Disable Slide Heater 18 ***** Select SSC Wash *****19 Rinse Slide With Reaction Buffer 20 Adjust Slide Volume With ReactionBuffer 21 Apply Coverslip 22 Rinse Slide With Reaction Buffer 23 AdjustSlide Volume With Reaction Buffer 24 Apply Coverslip 25 ***** ProcedureSynchronization ***** 26 Warm Slide to 37° C., and Incubate for 4Minutes 27 Rinse Slide With Reaction Buffer 28 Adjust Slide Volume WithReaction Buffer 29 Apply One Drop of I-VIEW INHIBITOR, Apply Coverslip,and Incubate for 4 Minutes 30 Rinse Slide With Reaction Buffer 31 AdjustSlide Volume With Reaction Buffer 32 Apply One Drop of [PROTEASE 1](Enzyme), Apply Coverslip, and Incubate for [8 Minutes] 33 Rinse SlideWith Reaction Buffer 34 Adjust Slide Volume With Reaction Buffer 35Apply Coverslip 36 Warm Slide to 37° C., and Incubate for 4 Minutes 37Rinse Slide With Reaction Buffer 38 Adjust Slide Volume With ReactionBuffer 39 Apply Coverslip 40 Apply One Drop of Antibody (e.g., clone3C6), and Incubate for [0 Hr 32 Min] 41 Rinse Slide With Reaction Buffer42 Adjust Slide Volume With Reaction Buffer 43 Apply Coverslip 44 WarmSlide to 37° C., and Incubate for 4 Minutes 45 Rinse Slide With ReactionBuffer 46 Adjust Slide Volume With Reaction Buffer 47 Apply One Drop ofI-VIEW BIOTIN Ig, Apply Coverslip, and Incubate for 8 Minutes 48 RinseSlide With Reaction Buffer 49 Adjust Slide Volume With Reaction Buffer50 Apply One Drop of I-VIEW SA-HRP, Apply Coverslip, and Incubate for 8Minutes 51 Rinse Slide With Reaction Buffer 52 Adjust Slide Volume WithReaction Buffer 53 Apply Coverslip 54 Rinse Slide With Reaction Buffer55 Adjust Slide Volume With Reaction Buffer 56 Apply One Drop of I-VIEWDAB and One Drop of I-VIEW H₂O₂, Apply Coverslip, Incubate for 8 Minutes57 Rinse Slide With Reaction Buffer 58 Adjust Slide Volume With ReactionBuffer 59 Apply One Drop of I-VIEW COPPER, Apply Coverslip, and Incubatefor 4 Minutes 60 Rinse Slide With Reaction Buffer 61 Adjust Slide VolumeWith Reaction Buffer 62 Apply One Drop of [HEMATOXYLIN II](Counterstain), Apply Coverslip, and Incubate for [4 Minutes] 63 RinseSlide With Reaction Buffer 64 Adjust Slide Volume With Reaction Buffer65 Apply Coverslip 66 Rinse Slide With Reaction Buffer 67 Adjust SlideVolume With Reaction Buffer 68 Apply One Drop of [BLUING REAGENT] (PostCounterstain), Apply Coverslip, and Incubate for [4 Minutes] 69 RinseSlide With Reaction Buffer 70 Apply Coverslip 71 Disable Slide Heater 72***** Select Optional Wash ***** 73 ***** Select SSC Wash ***** 74 *****Start Timed Steps ***** 75 Rinse Slide With Reaction Buffer

Example 3 Antibody Specific for EGFR Regulatory Domain EpitopeUnexpectedly does not Substantially Bind to Some EGFR-Positive Tissues

This Example demonstrates that RD-binding molecules, such as clone 5B7,exhibited differential binding to EGFR-positive tissues (as detected byan antibody specific for the EGFR external domain). As described in moredetail below, but without being limited to a single theory, thisdifferential binding is believed to be due to the differentialexpression of EGFR regulatory proteins (e.g., SOCS proteins like SOCS1or SOCS3) in EGFR-positive tissues. Such regulatory proteins, whendirectly associated with the EGFR regulatory domain, mask the epitopesof RD-binding molecules.

A. Normal Human Tissues

The staining by IHC of antibodies specific for the EGFR regulatorydomain (i.e., clone 5B7) and external domain (i.e., clone 3C6) in FFPE30 normal human tissues were compared. Tissue arrays were obtained fromUSBiomax (Igamsville, Md.; Cat. No. FDA801). Automated stainingprotocols as described in Example 2 were used to stain the tissue arrayson a BenchMark™ automated tissues stainer.

As shown in FIG. 5, extracellular-domain-specific clone 3C6 positivelystained skin, testis, tonsil, liver and placenta tissues, which isconsistent with known EGFR expression patterns in normal tissue. FIG. 5also shows that regulatory-domain-specific clone 5B7 reacted positivelywith normal skin, testis, and tonsil. However, surprisingly, thisantibody did not react with normal liver (see, e.g., FIG. 5B) and hadvariable reactivity with placental EGFR depending on the stage ofdevelopment of the placenta (Virchows Archiv A Pathol Anat., 420:385393, 1992).

B. Human NSCLC Tumors

Non-small cell lung cancer (NSCLC) cells are known to express EGFR inapproximately 75% of tumors. Regulatory-domain-specific clone 5B7 andexternal-domain-specific clone 3C6 were used to stain a cohort of NSCLCcases from three commercially available tissue micro arrays (Array LC801and Array LC819 (Biomax; Ijamsville, Md.) and Array IMT-305 (Imgenex(San Diego, Calif.)).

As shown in Table 3, Subpart A, clone 3C6 detected EGFR in 83% of thelung cases (as would be expected based on literature estimates of EGFRstaining in NSCLC) while clone 5B7 stained positively 65% of lungtumors. This corresponds to an 18.5% discordance between clone 3C6 andclone 5B7 with the latter exhibiting no staining in 38 cases that werepositive for clone 3C6 staining.

TABLE 3 Summary of NSCLC Immunohistochemistry Study 3C6 5B7 PositiveNegative Total Subpart A Positive 132 1 133 Negative 38 34 72 Total 17035 205 Subpart B Sensitivity 78% Specificity 97% Overall 81% Kappa 67%

As summarized in Table 3, Subpart B, the two antibodies each stainedpositively in 78% of cases (i.e., Sensitivity (132/[132+38]); the twoantibodies each stained negatively in 97% of cases (i.e., Specificity(34/[1+34]). The overall agreement was 81% ([132+43]/205). The Kappastatistic, which is another measure of agreement, can be interpreted asfollows: <0=No agreement, 0.0-0.19=Poor agreement, 0.20-0.39=Fairagreement, 0.40-0.59=Moderate agreement, 0.60-0.79═Substantial agreementand 0.80-1.00=Almost perfect agreement (Landis and Koch, Biometrics,33:159-174, 1977). The Kappa score for 3C6 versus 5B7 was 67% whichfalls into the substantial agreement category.

Particular examples demonstrating differences in the binding of EGFRregulatory-domain-specific clone 5B7 and EGFR external-domain-specificclone 3C6 to squamous cell carcinomas of the lung are shown in FIG. 6.Case 1 (left panel) showed equivalent staining of cells by the twoantibodies, which indicates (among other things) that there is not adifference in the general sensitivity of clone 5B7 as compared to clone3C6 when the epitope for each is accessible. Case 2 (right panel) showeddistinctly different staining between the two antibodies with clone 5B7being negative and clone 3C6 being 3+ positive (0-3+ scale).

The differential binding of clone 5B7 (specific for the EGFRintracellular regulatory domain) as compared to clone 3C6 (specific forthe EGFR extracellular domain) in normal and neoplastic tissues as shownin this Example strongly supports the belief that the clone 5B7 epitopewas accessible only in some tissues.

Example 4 Epitope Mapping of Monoclonal Antibody Clone 5B7

The epitope for the EGFR regulatory-domain-specific monoclonal antibody,clone 5B7, was mapped by peptide inhibition studies. Because the L2GPeptide was used as the immunogen and was used to screen for positiveclones, it was known that the 5B7 epitope must be within that 19-aminoacid sequence (see SEQ ID NO: 2). The L2G Peptide, a peptide containingthe 13 C-terminal amino acid residues of the L2G Peptide, and a peptidecontaining the six N-terminal amino acid residues of the L2G Peptideplus three additional N-terminal residues (i.e., QIS) corresponding tothe respective positions in the human EGFR sequence. The amino acidsequences of the subject peptides are shown in FIG. 7A. The peptideswere synthesized by Genemed Synthesis, Inc. (South San Francisco,Calif.).

A known EGFR-positive lung squamous cell carcinoma was chosen for thepeptide inhibition study. The 5B7 antibody was pre-incubated with eachpeptide for 1 hour at room temperature before application to the tissue.A 1000-fold molar excess of peptide compared to antibody was used.

As shown in FIG. 7, Peptide 1 resulted in partial inhibition of 5B7binding, which indicated that part of the epitope was contained withinPeptide 1. Peptide 2 resulted in complete inhibition of 5B7 binding,which indicated that the primary epitope was contained in Peptide 2.Logically, the three amino acids shared by Peptides 1 and 2 (i.e., QQD)must contain at least part of the full epitope. Peptide 3 alsocompletely inhibited 5B7 binding; thus, the tyrosine did notsignificantly contribute to the 5B7 epitope. An average epitope is onthe order of 7-12 contiguous amino acids; thus, the boxed residues inFIG. 7 represent a likely full-length 5B7 epitope with some possibilityfor an additional 1 to 5 residues at the C-terminal end.

Example 5 SOCS3 Knockout Unmasks Clone 5B7 Epitope

The L2G Peptide of the EGFR sequence is within the binding region forSOCS3 (Xia et al., J. Biol. Chem., 277(34):30716-23, 2002). Thus, it waspostulated that SOCS3 may be masking the 5B7 epitope in some tissues. Totest this hypothesis, livers from a hepatic-specific, SOCS3-knockoutmouse were obtained from the laboratory that developed the model (Ogataet al., Gastroenterology, 131(1): 179-93, 2006). Sections offormalin-fixed, paraffin-embedded livers from wild-type and SOCS3 micewere stained with clone 5B7 as described in Example 2.

As shown in the left two panels of FIG. 8, 5B7 failed to stain normalliver, which expresses SOCS3. In comparison, as shown in the right twopanels of FIG. 8, 5B7 positively stained the membranes of cells inlivers lacking SOCS3. These results indicate that the loss or absence ofSOCS3 allows for the binding of clone 5B7 to the regulatory domain ofEGFR.

SOCS3 is only one example of a regulatory molecule that directlyinteracts with EGFR. The results demonstrated herein are widelyapplicable to other interface-specific binding molecules that haveepitopes in the interface between two components of a molecular complex,such as between EGFR and its many regulatory proteins.

Example 6 EGFR RD-Binding Molecules, Such as Clone 5B7, Predict theResponse of NSCLC Cancer Patients to EGFR-Inhibitor Therapy

This Example demonstrates that a disclosed RD-binding molecule (e.g.,clone 5B7) predicts the response of NSCLC cancer patients toEGFR-inhibitor therapy (IRESSA™).

Tissue arrays containing biopsy samples from at least 100 NSCLC cancerpatients are obtained. Each patient is treated with IRESSA™(EGFR-inhibitor) therapy with a dosage of 250 mg/day given orally. Eachpatient has post-therapy follow-up for up to 5 years. Each biopsy sampleis fixed in 10% NBF and paraffin embedded. Five (5) micron sections ofeach biopsy sample are cut and arrayed on positively charged glassslides. The slides are stained with an RD-binding molecule (e.g., clone5B7) and an ED-binding molecule (e.g., clone 3C6) according to theprotocols in Example 2. The resulting stained array slides are scored bylight microscopy by a pathologist according to the following criteria:

Staining Intensity Report Result Score Microscope Observation Positive:Any IHC staining of 3+  Strong reactivity: Dark brown to black stainingis usually, but not tumor cell membranes above always, in a completemembrane pattern, producing a thick outline background level whether itis of the cell. Cytoplasmic reactivity may be absent or may be completeor incomplete moderately intense when membrane staining is very intense.circumferential staining in more Submembranous cytoplasmic accentuationmay be present. than 0% tumor cells 2.5 Intense reactivity: Shades ofbrown staining of medium darkness (intensity). Membranous reactivity isusually but not always complete, producing a circular outline of theneoplastic cell. Incomplete membrane reactivity of moderate intensity isalso considered 2+. The cytoplasmic reactivity is of weaker intensitythan the membrane reactivity. 2+  Moderate reactivity: Shades of brownstaining of intermediate darkness (intensity). Membranous reactivity isusually but not always complete, producing a circular outline of theneoplastic cell. Incomplete membrane reactivity of moderate intensity isalso considered 2+. The cytoplasmic reactivity is of weaker intensitythan the membrane reactivity. 1.5 Slight reactivity: Staining ofintermediate intensity that is membraneous. Cytoplasmic reactivity thatis uniform and involves all the cytoplasm may be present, but should notbe evaluated for positivity. 1+  Weak reactivity: Faint or light brownreactivity that is membranous. Cytoplasmic reactivity that is uniformand involves all the cytoplasm may be present, but should not beevaluated for positivity. Negative: Absence of membrane 0.5 Tracereactivity: Trace brown reactivity where membranous and staining abovebackground in all cytoplasmic localization is indeterminate. tumorcells. Presence of 0   No reactivity cytoplasmic in the absence ofmembrane staining.

The score for each case is recorded in a database comparing the scorefor each binding molecule (e.g., 5B7 or 3C6). The result of each case isassigned to 1 of the 4 categories described in Table 2. Approximately65% of cases are expected to fall into category 1, 19% in category 2,<1% in category 3 and 15% in category 4. Patient outcome is directlyrelated to the scoring category as indicated in Table 2 for an ID-basedtherapy. Patients in categories 1 and 3 will have an objective responseto IRESSA™ therapy and patients in categories 2 and 4 will notsignificantly respond to IRESSA™ therapy.

Example 7 EGFR RD-Binding Molecules, Such as Clone 5B7, Predict theResponse of NSCLC Cancer Patients to EGFR-Inhibitor Therapy

This Example demonstrates that a disclosed RD-binding molecule (e.g.,clone 5B7) predicts the response of NSCLC cancer patients toEGFR-inhibitor therapy (TARCEVA™).

Tissue arrays containing biopsy samples from at least 100 NSCLC cancerpatients are obtained. Each patient is treated with TARCEVA™ (EGFRinhibitor) therapy with a dosage of 150 mg/day given orally. Eachpatient has post-therapy follow-up for up to 5 years. Each biopsy sampleis fixed in 10% NBF and paraffin embedded. Five (5) micron sections ofeach biopsy sample are cut and arrayed on positively charged glassslides. The slides are stained with an RD-binding molecule (e.g., clone5B7) and an ED-binding molecule (e.g., clone 3C6) according to theprotocols in Example 2. The resulting stained array slides are scored bylight microscopy by a pathologist according to the following criteria:

Staining Intensity Report Result Score Microscope Observation Positive:Any IHC staining of 3+  Strong reactivity: Dark brown to black stainingis usually, but not tumor cell membranes above always, in a completemembrane pattern, producing a thick outline of background level whetherit is the cell. Cytoplasmic reactivity may be absent or may bemoderately complete or incomplete intense when membrane staining is veryintense. Submembranous circumferential staining in more cytoplasmicaccentuation may be present. than 0% tumor cells 2.5 Intense reactivity:Shades of brown staining of medium darkness (intensity). Membranousreactivity is usually but not always complete, producing a circularoutline of the neoplastic cell. Incomplete membrane reactivity ofmoderate intensity is also considered 2+. The cytoplasmic reactivity isof weaker intensity than the membrane reactivity. 2+  Moderatereactivity: Shades of brown staining of intermediate darkness(intensity). Membranous reactivity is usually but not always complete,producing a circular outline of the neoplastic cell. Incomplete membranereactivity of moderate intensity is also considered 2+. The cytoplasmicreactivity is of weaker intensity than the membrane reactivity. 1.5Slight reactivity: Staining of intermediate intensity that ismembraneous. Cytoplasmic reactivity that is uniform and involves all thecytoplasm may be present, but should not be evaluated for positivity.1+  Weak reactivity: Faint or light brown reactivity that is membranous.Cytoplasmic reactivity that is uniform and involves all the cytoplasmmay be present, but should not be evaluated for positivity. Negative:Absence of membrane 0.5 Trace reactivity: Trace brown reactivity wheremembranous and staining above background in all cytoplasmic localizationis indeterminate. tumor cells. Presence of 0   No reactivity cytoplasmicin the absence of membrane staining.

The score for each case is recorded in a database comparing the scorefor each binding molecule (e.g., 5B7 or 3C6). The result of each case isassigned to 1 of the 4 categories described in Table 2. Approximately65% of cases are expected to fall into category 1, 19% in category 2,<1% in category 3 and 15% in category 4. Patient outcome is directlyrelated to the scoring category as indicated in Table 2 for an ID-basedtherapy. Patients in categories 1 and 3 will have an objective responseto TARCEVA™ therapy and Patients in categories 2 and 4 will notsignificantly respond to TARCEVA™ therapy.

Example 8 EGFR RD-Binding Molecules, Such as Clone 5B7, Predict theResponse of Colorectal Cancer Patients to EGFR-Inhibitor Therapy

This Example demonstrates that a disclosed RD-binding molecule (e.g.,clone 5B7) predicts the response of colorectal cancer patients toEGFR-inhibitor therapy (ERBITUX™).

Tissue arrays containing biopsy samples from at least 100 colorectalcancer patients are obtained. Each patient is treated with ERBITUX™(EGFR inhibitor) therapy with a dosage of 400 mg/m² given i.v. Eachpatient has post-therapy follow-up for up to 5 years. Each biopsy sampleis fixed in 10% NBF and paraffin embedded. Five (5) micron sections ofeach biopsy sample are cut and arrayed on positively charged glassslides. The slides are stained with an RD-binding molecule (e.g., clone5B7) and an ED-binding molecule (e.g., clone 3C6) according to theprotocols in Example 2. The resulting stained array slides are scored bylight microscopy by a pathologist according to the following criteria:

Staining Intensity Report Result Score Microscope Observation Positive:Any IHC staining of 3+  Strong reactivity: Dark brown to black stainingis usually, but not tumor cell membranes above always, in a completemembrane pattern, producing a thick outline background level whether itis of the cell. Cytoplasmic reactivity may be absent or may be completeor incomplete moderately intense when membrane staining is very intense.circumferential staining in more Submembranous cytoplasmic accentuationmay be present. than 0% tumor cells 2.5 Intense reactivity: Shades ofbrown staining of medium darkness (intensity). Membranous reactivity isusually but not always complete, producing a circular outline of theneoplastic cell. Incomplete membrane reactivity of moderate intensity isalso considered 2+. The cytoplasmic reactivity is of weaker intensitythan the membrane reactivity. 2+  Moderate reactivity: Shades of brownstaining of intermediate darkness (intensity). Membranous reactivity isusually but not always complete, producing a circular outline of theneoplastic cell. Incomplete membrane reactivity of moderate intensity isalso considered 2+. The cytoplasmic reactivity is of weaker intensitythan the membrane reactivity. 1.5 Slight reactivity: Staining ofintermediate intensity that is membraneous. Cytoplasmic reactivity thatis uniform and involves all the cytoplasm may be present, but should notbe evaluated for positivity. 1+  Weak reactivity: Faint or light brownreactivity that is membranous. Cytoplasmic reactivity that is uniformand involves all the cytoplasm may be present, but should not beevaluated for positivity. Negative: Absence of membrane 0.5 Tracereactivity: Trace brown reactivity where membranous and staining abovebackground in all cytoplasmic localization is indeterminate. tumorcells. Presence of 0   No reactivity cytoplasmic in the absence ofmembrane staining.

The score for each case is recorded in a database comparing the scorefor each binding molecule (e.g., 5B7 or 3C6). The result of each case isassigned to 1 of the 4 categories described in Table 2. Approximately65% of cases are expected to fall into category 1, 19% in category 2,<1% in category 3 and 15% in category 4. Patient outcome is directlyrelated to the scoring category as indicated in Table 2 for an ED-basedtherapy. Patients in category 1 will have an objective response toERBITUX™ therapy, and patients in categories 2, 3 and 4 will notsignificantly respond to ERBITUX™ therapy.

Example 9 EGFR RD-Binding Molecules, Such as Clone 5B7, Predict theResponse of Colorectal Cancer Patients to EGFR-Inhibitor Therapy

This Example demonstrates that a disclosed RD-binding molecule (e.g.,clone 5B7) predicts the response of colorectal cancer patients toEGFR-inhibitor therapy (VECTIBIX™).

Tissue arrays containing biopsy samples from at least 100 colorectalcancer patients are obtained. Each patient is treated with VECTIBIX™(EGFR inhibitor) therapy with a dosage of 6 mg/kg given i.v. Eachpatient has post-therapy follow-up for up to 5 years. Each biopsy sampleis fixed in 10% NBF and paraffin embedded. Five (5) micron sections ofeach biopsy sample are cut and arrayed on positively charged glassslides. The slides are stained with an RD-binding molecule (e.g., clone5B7) and an ED-binding molecule (e.g., clone 3C6) according to theprotocols in Example 2. The resulting stained array slides are scored bylight microscopy by a pathologist according to the following criteria:

Staining Intensity Report Result Score Microscope Observation Positive:Any IHC staining of 3+  Strong reactivity: Dark brown to black stainingis usually, but not tumor cell membranes above always, in a completemembrane pattern, producing a thick outline background level whether itis of the cell. Cytoplasmic reactivity may be absent or may be completeor incomplete moderately intense when membrane staining is very intense.circumferential staining in more Submembranous cytoplasmic accentuationmay be present. than 0% tumor cells 2.5 Intense reactivity: Shades ofbrown staining of medium darkness (intensity). Membranous reactivity isusually but not always complete, producing a circular outline of theneoplastic cell. Incomplete membrane reactivity of moderate intensity isalso considered 2+. The cytoplasmic reactivity is of weaker intensitythan the membrane reactivity. 2+  Moderate reactivity: Shades of brownstaining of intermediate darkness (intensity). Membranous reactivity isusually but not always complete, producing a circular outline of theneoplastic cell. Incomplete membrane reactivity of moderate intensity isalso considered 2+. The cytoplasmic reactivity is of weaker intensitythan the membrane reactivity. 1.5 Slight reactivity: Staining ofintermediate intensity that is membraneous. Cytoplasmic reactivity thatis uniform and involves all the cytoplasm may be present, but should notbe evaluated for positivity. 1+  Weak reactivity: Faint or light brownreactivity that is membranous. Cytoplasmic reactivity that is uniformand involves all the cytoplasm may be present, but should not beevaluated for positivity. Negative: Absence of membrane 0.5 Tracereactivity: Trace brown reactivity where membranous and staining abovebackground in all cytoplasmic localization is indeterminate. tumorcells. Presence of 0   No reactivity cytoplasmic in the absence ofmembrane staining.

The score for each case is recorded in a database comparing the scorefor each binding molecule (e.g., 5B7 or 3C6). The result of each case isassigned to 1 of the 4 categories described in Table 2. Approximately65% of cases are expected to fall into category 1, 19% in category 2,<1% in category 3 and 15% in category 4. Patient outcome is directlyrelated to the scoring category as indicated in Table 2 for an ED-basedtherapy. Patients in category 1 will have an objective response toVECTIBIX™ therapy and, patients in categories 2, 3 and 4 will notsignificantly respond to VECTIBIX™ therapy.

Example 10 EGFR RD-Binding Molecules, Such as Clone 5B7, Predict theResponse of Breast Cancer Patients to EGFR-Inhibitor Therapy

This Example demonstrates that a disclosed RD-binding molecule (e.g.,clone 5B7) predicts the response of breast cancer patients toEGFR-inhibitor therapy or, more particularly, HER1 (EGFR)/HER2-inhibitortherapy (such as, lapatinib (TYKERB™)).

Tissue arrays containing biopsy samples from at least 100 breast cancerpatients are obtained. Each patient is treated with lapatinib (TYKERB™)(HER1 (EGFR)/HER2-inhibitor) with a dosage of 1250-1500 mg/day givenorally. Each patient has post-therapy follow-up for at least 20 months.Each biopsy sample is fixed in a standard fixative and paraffinembedded. Sections of each biopsy sample (e.g., 5 μm thick) are cut andarrayed on positively charged glass slides. The slides are stained withan RD-binding molecule (e.g., clone 5B7) and an ED-binding molecule(e.g., clone 3C6) according to the protocols in Example 2. The resultingstained array slides are scored by light microscopy by a pathologistaccording to the following criteria:

Staining Intensity Report Result Score Microscope Observation Positive:Any IHC staining of 3+  Strong reactivity: Dark brown to black stainingis usually, but not tumor cell membranes above always, in a completemembrane pattern, producing a thick outline background level whether itis of the cell. Cytoplasmic reactivity may be absent or may be completeor incomplete moderately intense when membrane staining is very intense.circumferential staining in more Submembranous cytoplasmic accentuationmay be present. than 0% tumor cells 2.5 Intense reactivity: Shades ofbrown staining of medium darkness (intensity). Membranous reactivity isusually but not always complete, producing a circular outline of theneoplastic cell. Incomplete membrane reactivity of moderate intensity isalso considered 2+. The cytoplasmic reactivity is of weaker intensitythan the membrane reactivity. 2+  Moderate reactivity: Shades of brownstaining of intermediate darkness (intensity). Membranous reactivity isusually but not always complete, producing a circular outline of theneoplastic cell. Incomplete membrane reactivity of moderate intensity isalso considered 2+. The cytoplasmic reactivity is of weaker intensitythan the membrane reactivity. 1.5 Slight reactivity: Staining ofintermediate intensity that is membraneous. Cytoplasmic reactivity thatis uniform and involves all the cytoplasm may be present, but should notbe evaluated for positivity. 1+  Weak reactivity: Faint or light brownreactivity that is membranous. Cytoplasmic reactivity that is uniformand involves all the cytoplasm may be present, but should not beevaluated for positivity. Negative: Absence of membrane 0.5 Tracereactivity: Trace brown reactivity where membranous and staining abovebackground in all cytoplasmic localization is indeterminate. tumorcells. Presence of 0   No reactivity cytoplasmic in the absence ofmembrane staining.

The score for each case is recorded in a database comparing the scorefor each binding molecule (e.g., 5B7 or 3C6). The result of each case isassigned to 1 of the 4 categories described in Table 2. Approximately20% of cases are expected to fall into category 1, 16% in category 2, 3%in category 3 and 61% in category 4. Patient outcome is directly relatedto the scoring category as indicated in Table 2 for an ID-based therapy.Patients in categories 1 and 3 will have an objective response tolapatinib (TYKERB™) therapy and Patients in categories 2 and 4 will notsignificantly respond to lapatinib (TYKERB™) therapy.

Example 11 EGFR RD-Binding Molecules, Such as Clone 5B7, Predict theResponse of Hepatocellular Carcinoma Cancer Patients to EGFR-InhibitorTherapy

This Example demonstrates that a disclosed RD-binding molecule (e.g.,clone 5B7) predicts the response of hepatocellular carcinoma (“HCC”)(such as, resectable HCC) cancer patients to EGFR-inhibitor therapy(IRESSA™).

Tissue arrays containing biopsy samples from at least 100 HCC cancerpatients are obtained (see, for example, samples collected in JS 0414,“A Pilot Study of Adjuvant Therapy of Gefitinib (Iressa, ZD1839) inPatients with Resectable Hepatocellular Carcinoma”, ClinicalTrials.govIdentifier No. NCT00228501). Each patient is treated with IRESSA™(EGFR-inhibitor) therapy with a dosage of 200-500 mg/day given orally.Each patient has post-therapy follow-up for at least 12 months. Eachbiopsy sample is fixed in a standard fixative (e.g., 10% NBF) andparaffin embedded. Sections of each biopsy sample (e.g., 5 μm thick) arecut and arrayed on positively charged glass slides. The slides arestained with an RD-binding molecule (e.g., clone 5B7) and an ED-bindingmolecule (e.g., clone 3C6) according to the protocols in Example 2. Theresulting stained array slides are scored by light microscopy by apathologist according to the following criteria:

Staining Intensity Report Result Score Microscope Observation Positive:Any IHC staining of 3+  Strong reactivity: Dark brown to black stainingis usually, but not tumor cell membranes above always, in a completemembrane pattern, producing a thick outline of background level whetherit is the cell. Cytoplasmic reactivity may be absent or may bemoderately complete or incomplete intense when membrane staining is veryintense. Submembranous circumferential staining in more cytoplasmicaccentuation may be present. than 0% tumor cells 2.5 Intense reactivity:Shades of brown staining of medium darkness (intensity). Membranousreactivity is usually but not always complete, producing a circularoutline of the neoplastic cell. Incomplete membrane reactivity ofmoderate intensity is also considered 2+. The cytoplasmic reactivity isof weaker intensity than the membrane reactivity. 2+  Moderatereactivity: Shades of brown staining of intermediate darkness(intensity). Membranous reactivity is usually but not always complete,producing a circular outline of the neoplastic cell. Incomplete membranereactivity of moderate intensity is also considered 2+. The cytoplasmicreactivity is of weaker intensity than the membrane reactivity. 1.5Slight reactivity: Staining of intermediate intensity that ismembraneous. Cytoplasmic reactivity that is uniform and involves all thecytoplasm may be present, but should not be evaluated for positivity.1+  Weak reactivity: Faint or light brown reactivity that is membranous.Cytoplasmic reactivity that is uniform and involves all the cytoplasmmay be present, but should not be evaluated for positivity. Negative:Absence of membrane 0.5 Trace reactivity: Trace brown reactivity wheremembranous and staining above background in all cytoplasmic localizationis indeterminate. tumor cells. Presence of 0   No reactivity cytoplasmicin the absence of membrane staining.

The score for each case is recorded in a database comparing the scorefor each binding molecule (e.g., 5B7 or 3C6). The result of each case isassigned to 1 of the 4 categories described in Table 2. Approximately60% of cases are expected to fall into category 1, 13% in category 2, 5%in category 3 and 22% in category 4. Patient outcome is directly relatedto the scoring category as indicated in Table 2 for an ID-based therapy.Patients in categories 1 and 3 will have an objective response toIRESSA™ therapy and patients in categories 2 and 4 will notsignificantly respond to IRESSA™ therapy.

Example 12 Clone 5B7 Status is a Clear Indicator of Lung CancerPrognosis

This Example demonstrates that clone 5B7 predicts the prognosis of lungcancer patients.

A tissue array containing lung biopsy samples from 109 Stage I or IINSCLC patients was obtained (a subset of the larger cohort described inOlaussen et al., New Engl. J. Med., 355(10):983-991, 2006). None of thepatients from whom the biopsies were obtained had been treated with anEGFR-based therapy (e.g., ERBITUX™, VECTIBIX™, IRESSA™, or TARCEVA™).Patient survival post-diagnosis was monitored on a continuing basis.Each biopsy sample was paraffin embedded, cancerous areas in the biopsywere identified, a core of the cancerous area removed, and placed in adonor array paraffin block. Three to five micron sections of the donorarray block were cut and mounted on glass slides. Slides containingserial sections of the donor array block were stained with clone 5B7 orclone 3C6 according to the protocols in Example 2. The resulting stainedslides are scored by light microscopy by a pathologist according to thefollowing criteria:

Staining Intensity Report Result Score Microscope Observation Positive:Any IHC staining of 3+  Strong reactivity: Dark brown to black stainingis usually, but not tumor cell membranes above always, in a completemembrane pattern, producing a thick outline of background level whetherit is the cell. Cytoplasmic reactivity may be absent or may bemoderately complete or incomplete intense when membrane staining is veryintense. Submembranous circumferential staining in more cytoplasmicaccentuation may be present. than 0% tumor cells 2.5 Intense reactivity:Shades of brown staining of medium darkness (intensity). Membranousreactivity is usually but not always complete, producing a circularoutline of the neoplastic cell. Incomplete membrane reactivity ofmoderate intensity is also considered 2+. The cytoplasmic reactivity isof weaker intensity than the membrane reactivity. 2+  Moderatereactivity: Shades of brown staining of intermediate darkness(intensity). Membranous reactivity is usually but not always complete,producing a circular outline of the neoplastic cell. Incomplete membranereactivity of moderate intensity is also considered 2+. The cytoplasmicreactivity is of weaker intensity than the membrane reactivity. 1.5Slight reactivity: Staining of intermediate intensity that ismembraneous. Cytoplasmic reactivity that is uniform and involves all thecytoplasm may be present, but should not be evaluated for positivity.1+  Weak reactivity: Faint or light brown reactivity that is membranous.Cytoplasmic reactivity that is uniform and involves all the cytoplasmmay be present, but should not be evaluated for positivity. Negative:Absence of membrane 0.5 Trace reactivity: Trace brown reactivity wheremembranous and staining above background in all cytoplasmic localizationis indeterminate. tumor cells. Presence of 0   No reactivity cytoplasmicin the absence of membrane staining.

The score for each biopsy sample and the associated follow-up is shownin the following table:

Months Sample 3C6 5B7 Months at at Number Score Score Recurrence DeathP003 2 2 None None P005 2.5 0.5 35 36 P006 0 2  6 36 P007 0 2 None NoneP008 0 1  5 25 P010 3 0.5 None None P011 2.5 0.5 42 57 P012 2.5 2 NoneNone P014 2 2.5 70 None P015 0 1 None None P016 1 1 None None P017 2.5 217 33 P018 2.5 2 None None P019 3 2 None None P020 0.5 1 None None P0222 1.5 None None P023 0 2 17 None P024 0 0.5 None None P025 0 2.5 NoneNone P026 0 2.5 16 94 P029 0.5 1 47 24 P030 0 2 None None P031 2 1.5 3670 P033 3 2 None None P037 0.5 4.5 None 96 P039 0 1 None None P041 0.51.5 None None P042 1 0 None None P044 1 1 None None P047 0 2  8 25 P0501.5 0 None None P051 2.5 0.5 None None P057 1 0 90 None P058 0 0.5 NoneNone P061 2 1 None None P063 1.5 3 None None P064 1.5 2.5 65 None P065 01.5 None None P066 0.5 0.5 None None P068 3 1 None None P070 1 1  5  7P071 0.5 3 15 18 P072 3 2 22 25 P073 1 0.5 68 None P074 2.5 0 None NoneP076 2 0.5 None None P078 3 1.5 None None P080 0.5 3 None None P161 0 0None None P162 2 2 None None P163 2 1 None None P165 1 1 None None P1670.5 1.5 None None P168 3 3 None None P169 2.5 2.5 None None P170 2 2.5None None P171 1 1 None None P172 0 0.5 19 28 P173 0 1 None None P174 22 None None P175 0.5 2 None None P176 0.5 0.5 None None P177 0.5 2 NoneNone P178 2 2 None None P179 3 2.5 None None P180 1.5 1 None None P1812.5 2.5 None None P182 3 3 None None P184 3 3 16 44 P187 2 2.5 None NoneP188 1 1 None None P189 0 1 17 None P190 3 3 17 44 P194 1.5 3 None NoneP197 0 1 None None P200 1 0.5 None None P202 3 3  8 20 P203 1.5 2  9 15P207 1 2.5 None None P209 2.5 2.5 None None P210 2 1 None None P211 2 3 4  5 P212 1 1.5 None None P213 3 3 29 None P214 2 2.5 None None P216 33 15 21 P217 3 3 19 None P219 2 2 None None P220 2 2 None None P221 00.5 None None P222 0 1  6 12 P223 0 0.5 None None P224 3 2.5 24 53 P2250 1 None None P226 1.5 2  5  9 P227 0.5 1.5 None None P228 1 2.5 NoneNone P230 1.5 1.5 None None P231 1 2 None None P232 0 0.5 None None

As shown in FIG. 9, clone 3C6 staining (whether negative or positive)has no correlation to NSCLC patient overall survivability while clone5B7 clearly delineates two populations. In particular, positive 5B7staining (score=1 or greater) identified NSCLC patients (n=80) havingpoor survivability, and negative 5B7 staining (score <1) identifiedNSCLC patients (n=20) with greater survivability. For example, as shownin FIG. 9B, approximately 82% of patients whose biopsy sample stainednegative for clone 5B7 were still surviving at 8.3 years post-diagnosis.In comparison, approximately 65% of patients whose biopsy sample stainedpositive for clone 5B7 were surviving at the same time point.

As shown in FIG. 10, clone 3C6 staining (whether negative or positive)has no correlation to NSCLC patient disease-free survival (DFS) whileclone 5B7 clearly delineates two populations. In particular, positive5B7 staining (score=1 or greater) identified NSCLC patients (n=80)having poor DFS, and negative 5B7 staining (score <1) identified NSCLCpatients (n=20) with greater DFS. For example, as shown in FIG. 10B,approximately 75% of patients whose biopsy sample stained negative forclone 5B7 were still surviving at 6 years post-diagnosis. In comparison,approximately 62% of patients whose biopsy sample stained positive forclone 5B7 were surviving at the same time point. The 5B7-positive and5B7-negative curves converge around 90 months post-diagnosis most likelydue to a statistical artifact cause by a decrease in the number of5B7-negative samples at that (and later) time points. It is expectedthat 5B7-negative NSCLC patients will continue to have a betterprognosis at 90 months and beyond when an even larger patient cohort isexamined.

This Example demonstrates that EGFR RD-binding molecules, such as clone5B7, predict the prognosis (e.g., overall survival and/or disease-freesurvival) of NSCLC patients (e.g., early stage NSCLC patients)independent of treatment.

Example 13 EGFR RD-Binding Molecules, Such as Clone 5B7, are Indicatorsof Colorectal Cancer Prognosis

This Example demonstrates that a disclosed RD-binding molecule (e.g.,clone 5B7) predicts the prognosis of colorectal cancer patients.

Tissue arrays containing biopsy samples from at least 100 colorectalcancer patients are obtained. Each patient preferably will not have beentreated with an EGFR-based therapy (e.g., ERBITUX™, VECTIBIX™, IRESSA™,or TARCEVA™). Each patient is followed for up to 5 years post-diagnosis.Each biopsy sample is fixed in 10% NBF and paraffin embedded. Five (5)micron sections of each biopsy sample are cut and arrayed on positivelycharged glass slides. The slides are stained with an RD-binding molecule(e.g., clone 5B7) and an ED-binding molecule (e.g., clone 3C6) accordingto the protocols in Example 2. The resulting stained array slides arescored by light microscopy by a pathologist according to the followingcriteria:

Staining Intensity Report Result Score Microscope Observation Positive:Any IHC staining of 3+  Strong reactivity: Dark brown to black stainingis usually, but not tumor cell membranes above always, in a completemembrane pattern, producing a thick outline of background level whetherit is the cell. Cytoplasmic reactivity may be absent or may bemoderately complete or incomplete intense when membrane staining is veryintense. Submembranous circumferential staining in more cytoplasmicaccentuation may be present. than 0% tumor cells 2.5 Intense reactivity:Shades of brown staining of medium darkness (intensity). Membranousreactivity is usually but not always complete, producing a circularoutline of the neoplastic cell. Incomplete membrane reactivity ofmoderate intensity is also considered 2+. The cytoplasmic reactivity isof weaker intensity than the membrane reactivity. 2+  Moderatereactivity: Shades of brown staining of intermediate darkness(intensity). Membranous reactivity is usually but not always complete,producing a circular outline of the neoplastic cell. Incomplete membranereactivity of moderate intensity is also considered 2+. The cytoplasmicreactivity is of weaker intensity than the membrane reactivity. 1.5Slight reactivity: Staining of intermediate intensity that ismembraneous. Cytoplasmic reactivity that is uniform and involves all thecytoplasm may be present, but should not be evaluated for positivity.1+  Weak reactivity: Faint or light brown reactivity that is membranous.Cytoplasmic reactivity that is uniform and involves all the cytoplasmmay be present, but should not be evaluated for positivity. Negative:Absence of membrane 0.5 Trace reactivity: Trace brown reactivity wheremembranous and staining above background in all cytoplasmic localizationis indeterminate. tumor cells. Presence of 0   No reactivity cytoplasmicin the absence of membrane staining.

The score for each case is recorded in a database comparing the scorefor each binding molecule (e.g., 5B7 and 3C6). The results of each casewill fall into one of the 4 categories described in Table 2.Approximately 65% of cases are expected to fall into category 1, 19% incategory 2, <1% in category 3 and 15% in category 4. Patient outcomewill be directly related to the scoring category as indicated in FIG. 3and Table 2. Patients in category 1 and 3 will have a poor prognosis,and patients in categories 2 and 4 will have a better prognosis.

Example 14 EGFR RD-Binding Molecules, Such as Clone 5B7, are Indicatorsof Head and Neck Cancer Prognosis

This Example demonstrates that a disclosed RD-binding molecule (e.g.,clone 5B7) predicts the prognosis of head and neck cancer patients.

Tissue arrays containing biopsy samples from at least 100 head and neckcancer patients are obtained. Each patient preferably will not have beentreated with an EGFR-based therapy (e.g., ERBITUX™, VECTIBIX™, IRESSA™,or TARCEVA™). Each patient is followed for up to 5 years post-diagnosis.Each biopsy sample is fixed in 10% NBF and paraffin embedded. Five (5)micron sections of each biopsy sample are cut and arrayed on positivelycharged glass slides. The slides are stained with an RD-binding molecule(e.g., clone 5B7) and an ED-binding molecule (e.g., clone 3C6) accordingto the protocols in Example 2. The resulting stained array slides arescored by light microscopy by a pathologist according to the followingcriteria:

Staining Intensity Report Result Score Microscope Observation Positive:Any IHC staining of 3+  Strong reactivity: Dark brown to black stainingis usually, but not tumor cell membranes above always, in a completemembrane pattern, producing a thick outline of background level whetherit is the cell. Cytoplasmic reactivity may be absent or may bemoderately complete or incomplete intense when membrane staining is veryintense. Submembranous circumferential staining in more cytoplasmicaccentuation may be present. than 0% tumor cells 2.5 Intense reactivity:Shades of brown staining of medium darkness (intensity). Membranousreactivity is usually but not always complete, producing a circularoutline of the neoplastic cell. Incomplete membrane reactivity ofmoderate intensity is also considered 2+. The cytoplasmic reactivity isof weaker intensity than the membrane reactivity. 2+  Moderatereactivity: Shades of brown staining of intermediate darkness(intensity). Membranous reactivity is usually but not always complete,producing a circular outline of the neoplastic cell. Incomplete membranereactivity of moderate intensity is also considered 2+. The cytoplasmicreactivity is of weaker intensity than the membrane reactivity. 1.5Slight reactivity: Staining of intermediate intensity that ismembraneous. Cytoplasmic reactivity that is uniform and involves all thecytoplasm may be present, but should not be evaluated for positivity.1+  Weak reactivity: Faint or light brown reactivity that is membranous.Cytoplasmic reactivity that is uniform and involves all the cytoplasmmay be present, but should not be evaluated for positivity. Negative:Absence of membrane 0.5 Trace reactivity: Trace brown reactivity wheremembranous and staining above background in all cytoplasmic localizationis indeterminate. tumor cells. Presence of 0   No reactivity cytoplasmicin the absence of membrane staining.

The score for each case is recorded in a database comparing the scorefor each binding molecule (e.g., 5B7 and 3C6). The results of each casewill fall into one of the 4 categories described in Table 2.Approximately 65% of cases are expected to fall into category 1, 19% incategory 2, <1% in category 3 and 15% in category 4. Patient outcomewill be directly related to the scoring category as indicated in FIG. 3and Table 2. Patients in category 1 and 3 will have a poor prognosis,and patients in categories 2 and 4 will have a better prognosis.

Example 15 EGFR RD-Binding Molecules, Such as Clone 5B7, are Indicatorsof Gastric Cancer Prognosis

This Example demonstrates that a disclosed RD-binding molecule (e.g.,clone 5B7) predicts the prognosis of gastric cancer patients.

Tissue arrays containing biopsy samples from at least 100 gastric cancerpatients are obtained. Each patient preferably will not have beentreated with an EGFR-based therapy (e.g., ERBITUX™, VECTIBIX™, IRESSA™,or TARCEVA™). Each patient is followed for up to 5 years post-diagnosis.Each biopsy sample is fixed in 10% NBF and paraffin embedded. Five (5)micron sections of each biopsy sample are cut and arrayed on positivelycharged glass slides. The slides are stained with an RD-binding molecule(e.g., clone 5B7) and an ED-binding molecule (e.g., clone 3C6) accordingto the protocols in Example 2. The resulting stained array slides arescored by light microscopy by a pathologist according to the followingcriteria:

Staining Intensity Report Result Score Microscope Observation Positive:Any IHC staining of 3+  Strong reactivity: Dark brown to black stainingis usually, but not tumor cell membranes above always, in a completemembrane pattern, producing a thick outline background level whether itis of the cell. Cytoplasmic reactivity may be absent or may be completeor incomplete moderately intense when membrane staining is very intense.circumferential staining in more Submembranous cytoplasmic accentuationmay be present. than 0% tumor cells 2.5 Intense reactivity: Shades ofbrown staining of medium darkness (intensity). Membranous reactivity isusually but not always complete, producing a circular outline of theneoplastic cell. Incomplete membrane reactivity of moderate intensity isalso considered 2+. The cytoplasmic reactivity is of weaker intensitythan the membrane reactivity. 2+  Moderate reactivity: Shades of brownstaining of intermediate darkness (intensity). Membranous reactivity isusually but not always complete, producing a circular outline of theneoplastic cell. Incomplete membrane reactivity of moderate intensity isalso considered 2+. The cytoplasmic reactivity is of weaker intensitythan the membrane reactivity. 1.5 Slight reactivity: Staining ofintermediate intensity that is membraneous. Cytoplasmic reactivity thatis uniform and involves all the cytoplasm may be present, but should notbe evaluated for positivity. 1+  Weak reactivity: Faint or light brownreactivity that is membranous. Cytoplasmic reactivity that is uniformand involves all the cytoplasm may be present, but should not beevaluated for positivity. Negative: Absence of membrane 0.5 Tracereactivity: Trace brown reactivity where membranous and staining abovebackground in all cytoplasmic localization is indeterminate. tumorcells. Presence of 0   No reactivity cytoplasmic in the absence ofmembrane staining.

The score for each case is recorded in a database comparing the scorefor each binding molecule (e.g., 5B7 and 3C6). The results of each casewill fall into one of the 4 categories described in Table 2.Approximately 65% of cases are expected to fall into category 1, 19% incategory 2, <1% in category 3 and 15% in category 4. Patient outcomewill be directly related to the scoring category as indicated in FIG. 3and Table 2. Patients in category 1 and 3 will have a poor prognosis,and patients in categories 2 and 4 will have a better prognosis.

Example 16 EGFR RD-Binding Molecules, Such as Clone 5B7, are Indicatorsof Glioblastoma Cancer Prognosis

This Example demonstrates that a disclosed RD-binding molecule (e.g.,clone 5B7) predicts the prognosis of glioblastoma cancer patients.

Tissue arrays containing biopsy samples from at least 100 glioblastomacancer patients are obtained. Each patient preferably will not have beentreated with an EGFR-based therapy (e.g., ERBITUX™, VECTIBIX™, IRESSA™,or TARCEVA™). Each patient is followed for up to 5 years post-diagnosis.Each biopsy sample is fixed in 10% NBF and paraffin embedded. Five (5)micron sections of each biopsy sample are cut and arrayed on positivelycharged glass slides. The slides are stained with an RD-binding molecule(e.g., clone 5B7) and an ED-binding molecule (e.g., clone 3C6) accordingto the protocols in Example 2. The resulting stained array slides arescored by light microscopy by a pathologist according to the followingcriteria:

Staining Intensity Report Result Score Microscope Observation Positive:Any IHC staining of 3+  Strong reactivity: Dark brown to black stainingis usually, but not tumor cell membranes above always, in a completemembrane pattern, producing a thick outline of background level whetherit is the cell. Cytoplasmic reactivity may be absent or may bemoderately complete or incomplete intense when membrane staining is veryintense. Submembranous circumferential staining in more cytoplasmicaccentuation may be present. than 0% tumor cells 2.5 Intense reactivity:Shades of brown staining of medium darkness (intensity). Membranousreactivity is usually but not always complete, producing a circularoutline of the neoplastic cell. Incomplete membrane reactivity ofmoderate intensity is also considered 2+. The cytoplasmic reactivity isof weaker intensity than the membrane reactivity. 2+  Moderatereactivity: Shades of brown staining of intermediate darkness(intensity). Membranous reactivity is usually but not always complete,producing a circular outline of the neoplastic cell. Incomplete membranereactivity of moderate intensity is also considered 2+. The cytoplasmicreactivity is of weaker intensity than the membrane reactivity. 1.5Slight reactivity: Staining of intermediate intensity that ismembraneous. Cytoplasmic reactivity that is uniform and involves all thecytoplasm may be present, but should not be evaluated for positivity.1+  Weak reactivity: Faint or light brown reactivity that is membranous.Cytoplasmic reactivity that is uniform and involves all the cytoplasmmay be present, but should not be evaluated for positivity. Negative:Absence of membrane 0.5 Trace reactivity: Trace brown reactivity wheremembranous and staining above background in all cytoplasmic localizationis indeterminate. tumor cells. Presence of 0   No reactivity cytoplasmicin the absence of membrane staining.

The score for each case is recorded in a database comparing the scorefor each binding molecule (e.g., 5B7 and 3C6). The results of each casewill fall into one of the 4 categories described in Table 2.Approximately 65% of cases are expected to fall into category 1, 19% incategory 2, <1% in category 3 and 15% in category 4. Patient outcomewill be directly related to the scoring category as indicated in FIG. 3and Table 2. Patients in category 1 and 3 will have a poor prognosis,and patients in categories 2 and 4 will have a better prognosis.

Example 17 EGFR RD-Binding Molecules, Such as Clone 5B7, are Indicatorsof Hepatocellular Carcinoma Prognosis

This Example demonstrates that a disclosed RD-binding molecule (e.g.,clone 5B7) predicts the prognosis of HCC (such as, resectable HCC)cancer patients Tissue arrays containing biopsy samples from at least100 HCC cancer patients are obtained (see, for example, control arm ofsamples collected in JS 0414, “A Pilot Study of Adjuvant Therapy ofGefitinib (Iressa, ZD1839) in Patients with Resectable HepatocellularCarcinoma”, ClinicalTrials.gov Identifier No. NCT00228501). Each patientpreferably will not have been treated with an EGFR-based therapy (e.g.,ERBITUX™, VECTIBIX™, IRESSA™, or TARCEVA™). Each patient is followed forup to 5 years post-diagnosis. Each biopsy sample is fixed in a standardfixative (e.g., 10% NBF) and paraffin embedded. Sections of each biopsysample (e.g., 5 μm thick) are cut and arrayed on positively chargedglass slides. The slides are stained with an RD-binding molecule (e.g.,clone 5B7) and an ED-binding molecule (e.g., clone 3C6) according to theprotocols in Example 2. The resulting stained array slides are scored bylight microscopy by a pathologist according to the following criteria:

Staining Intensity Report Result Score Microscope Observation Positive:Any IHC staining of 3+  Strong reactivity: Dark brown to black stainingis usually, but not tumor cell membranes above always, in a completemembrane pattern, producing a thick outline of background level whetherit is the cell. Cytoplasmic reactivity may be absent or may bemoderately complete or incomplete intense when membrane staining is veryintense. Submembranous circumferential staining in more cytoplasmicaccentuation may be present. than 0% tumor cells 2.5 Intense reactivity:Shades of brown staining of medium darkness (intensity). Membranousreactivity is usually but not always complete, producing a circularoutline of the neoplastic cell. Incomplete membrane reactivity ofmoderate intensity is also considered 2+. The cytoplasmic reactivity isof weaker intensity than the membrane reactivity. 2+  Moderatereactivity: Shades of brown staining of intermediate darkness(intensity). Membranous reactivity is usually but not always complete,producing a circular outline of the neoplastic cell. Incomplete membranereactivity of moderate intensity is also considered 2+. The cytoplasmicreactivity is of weaker intensity than the membrane reactivity. 1.5Slight reactivity: Staining of intermediate intensity that ismembraneous. Cytoplasmic reactivity that is uniform and involves all thecytoplasm may be present, but should not be evaluated for positivity.1+  Weak reactivity: Faint or light brown reactivity that is membranous.Cytoplasmic reactivity that is uniform and involves all the cytoplasmmay be present, but should not be evaluated for positivity. Negative:Absence of membrane 0.5 Trace reactivity: Trace brown reactivity wheremembranous and staining above background in all cytoplasmic localizationis indeterminate. tumor cells. Presence of 0   No reactivity cytoplasmicin the absence of membrane staining.

The score for each case is recorded in a database comparing the scorefor each binding molecule (e.g., 5B7 and 3C6). The results of each casewill fall into one of the 4 categories described in Table 2.Approximately 60% of cases are expected to fall into category 1, 13% incategory 2, 5% in category 3 and 22% in category 4. Patient outcome willbe directly related to the scoring category as indicated in FIG. 3 andTable 2. Patients in category 1 and 3 will have a poor prognosis, andpatients in categories 2 and 4 will have a better prognosis.

While this disclosure has been described with an emphasis uponparticular embodiments, it will be obvious to those of ordinary skill inthe art that variations of the particular embodiments may be used and itis intended that the disclosure may be practiced otherwise than asspecifically described herein. Accordingly, this disclosure includes allmodifications encompassed within the spirit and scope of the disclosureas defined by the following claims:

1. An isolated peptide consisting of amino acid residues 1167-1185 ofSEQ ID NO: 1 or an immunogenic fragment of said peptide.
 2. Acomposition comprising an epidermal growth factor receptor (EGFR)regulatory domain (RD)-binding molecule the binding of which to EGFR iscompetitively inhibited by the peptide of claim
 1. 3. The composition ofclaim 2, wherein the RD-binding molecule is an antibody.
 4. Thecomposition of claim 3, wherein the antibody is a monoclonal antibody.5. A composition comprising an antibody that specifically binds to thepeptide of claim
 1. 6. A composition comprising an EGFR RD-bindingmolecule, the binding of which to EGFR is competitively inhibited by aSuppressor of Cytokine Signaling (SOCS) protein.
 7. The composition ofclaim 6, wherein the RD-binding molecule is an antibody.
 8. Acomposition comprising a RD-binding molecule that specifically binds toresidues 1138-1196 of SEQ ID NO: 1 or a SOCS-protein-binding fragmentthereof
 9. A method of producing an EGFR-specific antibody, comprisingimmunizing a non-human mammal with an immunogen comprising a carrierprotein and the peptide of claim
 1. 10. A method of predicting theresponse of a neoplasm to an EGFR inhibitor, comprising: detecting in abiological sample, which comprises one or more neoplastic cells, thespecific binding of the antibody in the composition of claim 5 to one ormore of the neoplastic cells; wherein specific binding of the antibodyto one or more of the neoplastic cells indicates that the neoplasticcells will respond to an EGFR inhibitor.
 11. The method of claim 10,wherein the neoplastic cell response is slowed growth.
 12. The method ofclaim 10, wherein the neoplastic cell response is apoptosis.
 13. Themethod of claim 10, wherein the biological sample is mounted on amicroscope slide.
 14. The method of claim 13, wherein the biologicalsample is a tissue section.
 15. The method of claim 14, wherein thetissue section is formalin fixed and paraffin embedded.
 16. The methodof claim 10, wherein the biological sample is a neoplastic tissue.
 17. Amethod for predicting whether a candidate for treatment with an EGFRinhibitor is likely to respond to such treatment, comprising: detectingin a biological sample from a candidate for treatment with an EGFRinhibitor, which biological sample comprises one or more neoplasticcells, the specific binding of the antibody in the composition of claim5 to one or more of the neoplastic cells; wherein specific binding ofthe antibody to one or more of the neoplastic cells indicates that thecandidate is likely to respond to treatment with an EGFR inhibitor. 18.The method of claim 17, wherein the biological sample is mounted on amicroscope slide.
 19. The method of claim 18, wherein the biologicalsample is formalin fixed and paraffin embedded.
 20. The method of claim18, wherein the biological sample is a neoplastic tissue.
 21. The methodof claim 18, wherein the specific binding of the antibody to at least10% of the neoplastic cells in the biological sample indicates that thecandidate is likely to respond to treatment with an EGFR inhibitor. 22.A method of predicting the response of a neoplasm to an EGFR inhibitor,comprising: detecting in a biological sample comprising one or moreEGFR-positive neoplastic cells substantially no specific binding of theantibody in the composition of claim 5 to the one or more EGFR-positiveneoplastic cells; wherein substantially no specific binding of theantibody to the EGFR-positive neoplastic cells indicates that theneoplastic cells will not substantially respond to an EGFR inhibitor.23. The method of claim 22, further comprising detecting in a controlbiological material the specific binding of the antibody to EGFR. 24.The method of claim 22, further comprising detecting in the biologicalsample specific binding of a second antibody specific for the EGFRexternal domain.
 25. A method of predicting the response of a neoplasmto EGFR inhibitor administration, comprising: detecting EGFR expressionin a first sample of a biological material comprising one or moreneoplastic cells; and detecting in a second sample of the biologicalmaterial substantially no specific binding to EGFR of the antibody inthe composition of claim 5; wherein detecting EGFR expression in thefirst sample and substantially no specific binding to EGFR of theantibody indicates that the neoplasm is likely to respond to EGFRinhibitor administration.
 26. The method of claim 25, further comprisingdetecting in a control biological material the specific binding of theantibody to EGFR.
 27. A method for predicting prognosis of a neoplasticdisease, comprising: detecting in a biological sample from a patienthaving a neoplastic disease the specific binding of the antibody in thecomposition of claim 5 to one or more EGFR-positive neoplastic cells inthe biological sample; wherein the specific binding of the antibody inthe one or more EGFR-positive neoplastic cells predicts a poor prognosisof the neoplastic disease in the patient.
 28. The method of claim 27,wherein the antibody specifically binds to at least 10% of theEGFR-positive neoplastic cells in the biological sample.
 29. A methodfor predicting prognosis of a neoplastic disease, comprising: detectingin a biological sample from a patient having a neoplastic disease thespecific binding of the antibody in the composition of claim 5 to one ormore EGFR-positive neoplastic cells in the biological sample; whereinsubstantially no specific binding of the antibody in the one or moreEGFR-positive neoplastic cells predicts a good prognosis of theneoplastic disease in the patient.
 30. An immunostaining methodcomprising, contacting a biological sample, comprising one or morecells, with the antibody in the composition of claim 5, and detectingthe specific binding of the antibody to an antigen in the one or morecells.
 31. The method of claim 30, wherein the biological sample ismounted on a microscope slide.
 32. The method of claim 31, wherein thebiological sample is formalin fixed and paraffin embedded.
 33. Themethod of claim 30, wherein the antigen is EGFR.
 34. A method ofdetecting a direct interaction between EGFR and an EGFR regulatoryprotein, comprising: contacting a biological sample, comprising one ormore EGFR-positive cells, with the antibody in the composition of claim5, and detecting the specific binding of the antibody to the one or moreEGFR-positive cells, wherein the specific binding of the antibody to theone or more EGFR-positive cells detects that EGFR is not significantlyinteracting with an EGFR regulatory protein, wherein an interactionbetween EGFR and the EGFR regulatory protein masks the epitope of theantibody.
 35. A method of detecting a direct interaction between EGFRand an EGFR regulatory protein, comprising: contacting a biologicalsample, comprising one or more EGFR-positive cells, with the antibody inthe composition of claim 5, and detecting the specific binding of theantibody to the one or more EGFR-positive cells, wherein substantiallyno specific binding of the antibody to the one or more EGFR-positivecells detects that EGFR is interacting with an EGFR regulatory protein,wherein an interaction between EGFR and the EGFR regulatory proteinmasks the epitope of the antibody.